Kits and methods for detecting methylated dna

ABSTRACT

The present invention relates to an in vitro method for detecting methylated DNA comprising (a) coating a container with a polypeptide capable of binding methylated DNA; (b) contacting said polypeptide with a sample comprising methylated and/or unmethylated DNA; and (c) detecting the binding of said polypeptide to methylated DNA. In a preferred embodiment, said method further comprises step (d) analyzing the detected methylated DNA by sequencing. Another aspect of the present invention is a kit for detecting methylated DNA according to the methods of the invention comprising (a) a polypeptide capable of binding methylated DNA; (b) a container which can be coated with said polypeptide; (c) means for coating said container; and (d) means for detecting methylated DNA.

The present invention relates to an in vitro method for detectingmethylated DNA comprising (a) coating a container with a polypeptidecapable of binding methylated DNA; (b) contacting said polypeptide witha sample comprising methylated and/or unmethylated DNA; and (c)detecting the binding of said polypeptide to methylated DNA. In apreferred embodiment, said method further comprises step (d) analyzingthe detected methylated DNA by sequencing. Another aspect of the presentinvention is a kit for detecting methylated DNA according to the methodsof the invention comprising (a) a polypeptide capable of bindingmethylated DNA; (b) a container which can be coated with saidpolypeptide; (c) means for coating said container; and (d) means fordetecting methylated DNA.

The information to make the cells of all living organisms is containedin their DNA. DNA is made from 4 bases abbreviated as G, A, T, and C,and is built like a very long ladder with pairs of these letter makingup each of the “rungs” of the ladder. The letter G pairs with C and Awith T. Strings of these pairs store information like a coded message,with the information to make specific molecules grouped into regionscalled genes. Every cell of diploid animals contains two copies of everygene, with one copy of each gene coming from the mother and one copyfrom the father. (The only exceptions to this rule are genes onchromosomes that determine whether organisms develop as a “male” or a“female”.)

DNA Methylation and Gene Regulation

Apart from the four bases—adenine, guanine, cytosine and thymine—that“spell” our genome, there also is a fifth base which is produced by themodification of the post-replicative DNA. DNA methyl transferases(DNMTs) can catalyse the transfer of a methyl group from the methyldonor S-adenosylmethionine to the cytosine ring, and thereby produce thebase 5-methylcytosine. Specific cytosine residues are modified inmammals, which precede a guanosine residue in the DNA sequence (CpGdinucleotide) (Singal, Blood 93 (1999), 4059-4070); Robertson, Nat. Rev.Genet. 1 (2000), 11-19; Ng, Curr. Opin. Genet. Dev. (2000), 158-163;Razin, EMBO J. 17 (1998), 4905-4908). The methylation of CpGdinucleotides generally correlates with stable transcriptionalrepression and presumably leads to the fact that large parts of thenon-coding genome and potentially harmful sequences such as transposons,repeats or viral inserts are not transcribed. It is interesting that CpGdinucleotides are very unevenly distributed in the genome (Singal(1999), loc. cit., Robertson (2000), loc. cit., Ng (2000), loc. cit.,Razin (1998), loc. cit.). A large part of the genome contains much fewerCpGs than is statistically expected. This is presumably due to the factthat 5-methylcytosine deaminates comparatively easily to thymidine,which, in the course of evolution, leads to a relative decrease in thenumber of CpG dinucleotides. There are, however, again and again, largernumbers of CpGs distributed within the genome, so-called CpG islands.These regions often contain transcription initiation points and genepromoters and are generally not methylated in contrast to the CpGs whichare not associated with CpG islands. In normal cells, the methylation ofCpG islands has been observed only in exceptional cases such as theinactivation of the second copy of the x-chromosome in female cells andthe parental imprinting genome (Singal (1999), loc. cit., Robertson(2000), loc. cit., Ng (2000), loc. cit., Razin (1998), loc. cit.).

Regulation of DNA Methylation

It is only partly understood how DNA methylation patterns areestablished in the course of the embryogenesis and how the CpGmethylation is maintained and regulated in the genome (Singal (1999),loc. cit., Ng (2000), loc. cit., Razin (1998), loc. cit.). In mammalspecies, there are three DNA methyl transferases known (DNMT1, 3a and3b) which catalyse the DNA methylation process. The corresponding sharethat each DNMT contributes to the maintenance and regulation of the CpGmethylation must, however, still be clarified. Yet, all three enzymesare obviously essential to embryogenesis, the corresponding knockoutmice die in utero or shortly after birth (Bestor, Hum. Mol. Genet. 9(2000), 2395-2402; El Osta, Bioessays 25 (2003), 1071-1084). In themeantime, the connection between DNA methylation, modifications of thechromatin structure and certain histone modifications has been shownseveral times. The methylation of DNA mostly correlates with histonedeacetylation and methylation of the lysine 9 residue at histone H3(Sims, Trends Genet. 19 (2003), 629-639, Fahrner, Cancer Res. 62 (2002),7213-7218). Accordingly, DNMTs are associated with histone acetylases(HDACs) or co-repressor complexes. It is also hardly known how methylgroups are removed from CpG residues. In proliferating cells, the DNAmethylation can probably also take place passively during replication.There are, however, also examples of DNA demethylation in post-mitototiccells which can be explained by the existence of an active, yet unknowndemethylase (Wolffe, Proc. Natl. Acad. Sci. 96 (1999), 5894-5896).

CpG Methylation and Gene Silencing

Methylation of promoters (but not of non-regulating sequences)correlates with stable, transcriptional repression (Singal (1999), loc.cit., Ng (2000), loc. cit., Razin (1998), loc. cit.). The repressiveproperties of 5-methylcytosine can be mediated by two mechanisms.Firstly, the DNA methylation can directly impair the binding oftranscription factors. The second possibility, which is likely to beresponsible for the largest part of repression, is the recruitment ofmethyl-CpG-binding proteins (MBPs) (Ballestar, Eur. J. Biochem. 268(2001), 1-6). MBPs such as MECP2 or MBD2 (a component of the MeCP1complex) are accompanied by co-repressor complexes and HDACs which havea repressive effect and are responsible for the formation of densechromatin structures inaccessible to transcription factors(heterochromatin) (Ballestar (2001), loc. cit.).

Epigenetic Changes in Tumorigenesis

It keeps becoming clearer that the formation of tumours is supported notonly by genetic lesions (e.g. mutations or translocations) but also byepigenetic changes. An abnormal chromatin structure or DNA methylationcan influence the transcriptional status of oncogenes or tumoursuppressor genes and can promote tumour growth. Changes in the DNAmethylation include either the loss of methylation in normallymethylated sequences (hypomethylation) or the methylation of normallyunmethylated sequences (hypermethylation) (Roberston (2000), loc. cit.,Herman, N. Engl. J. Med. 349 (2003), 2042-2054; Momparler, Oncogene 22(2003), 6479-6483; Esteller, Science 297 (2002), 1807-1808; Plass, Hum.Mol. Genet. 11 (2002), 2479-2488).

Hypomethylation

A global DNA hypomethylation has been described for almost all kinds oftumours. In tumour tissue, the content in 5-methylcytosine is reducedcompared to normal tissue with the major share of demethylation eventsbeing found in repetitive satellite sequences or in centromer regions ofthe chromosomes. However, in single cases, the demethylation andactivation of proto-oncogenes such as, e.g., bcl-2 or c-myc have alsobeen described (Costello, J. Med. Genet. 38 (2001), 285-303).

Hypermethylation of CpG Islands

CpG islands in general exert gene regulatory functions. This is why achange in the status of methylation correlates mostly directly with achange in the transcriptional activity of the locus concerned (Robertson(1999); Herman (2003); Esteller (2002); Momparler (2003); Plass (2002),all loc. cit.). Most CpG islands are present in unmethylated form innormal cells. In certain situations, CpG islands can, however, also bemethylated in gene regulatory events. The majority of CpG islands of theinactivated X-chromosome of a female cell are, for example, methylated(Goto, Microbiol. Mol. Biol. Rev. 62 (1998), 362-378). CpG islands canbe methylated also in the course of normal aging processes (Issa, Clin.Immunol. 109 (2003), 103-108).

It is in particular in tumours that CpG islands which are normally notmethylated can be present in a hypermethylated form. In many cases,genes affected by the hypermethylation encode proteins which counteractthe growth of a tumour such as, e.g., tumour suppressor genes. Thefollowing Table lists examples of genes for which it could be shown thatthey can be inactivated in tumours through the epigenetic mechanism ofhypermethylation.

TABLE Hypermethylated genes in tumours (examples) chromo- gene somefunction cell p16 9p21 cycline-dependent kinase inhibitor cycle p15 9p21cycline-dependent kinase inhibitor control Rb 13q14 cell cycleinhibition p73 1p36 p53-like protein DNA MLH1 3p21 DNA mismatch repairprotein repair GSTPI 11q13 inhibitor of oxidative DNA damage O6-MGMT10q26 DNA methyltransferase BRCA1 17q21 DNA repair protein apoptosisTMS-1/ASC 16p12-p11 adaptor for caspase 1 caspase 8 2q33-q34 PCDinitiator (Fas, Trail, TNF, . . .) DAPK1 9q34 PCD by IFNγ invasion/E-cadherin 16q22 adhesion molecule architec- VHL 3p26-p25angiogenesis-promoting protein ture TIMP-3 22q12-q13 metalloproteinaseinhibitor THBS1 15q15 angiogenesis inhibitor growth ER-α 6q25 estrogenreceptor factor RAR-β 3p24 retinoic acid receptor response SOCS-1 16p13neg. regulator in the JAK/STAT signal path

Reasons for the tumour-specific hypermethylation are almost unknown.Interestingly, certain kinds of tumours seem to have their ownhypermethylation profiles. It could be shown in larger comparativestudies that hypermethylation is not evenly distributed but that itoccurs depending on the tumour. In cases of leukaemia, mostly othergenes are hypermethylated compared to, for instance, colon carcinomas orgliomas. Thus, hypermethylation could be useful for classifying tumours(Esteller, Cancer Res. 61 (2001), 3225-3229; Costello, Nat. Genet. 24(2000), 132-138).

In many cases, hypermethylation is also combined with an increasedactivity of HDACs. After treatment with demethylating substances (e.g.5-azacytidine), many methylated genes could only be reactivated afteralso using HDAC inhibitors (such as, e.g., trichostatin A (TSA))(Suzuki, Nat. Genet. 31 (2002), 141-149; Ghoshal, Mol. Cell. Biol. 22(2002), 8302-8319; Kalebic, Ann. N.Y. Acad. Sci. 983 (2003), 278-285).

Most analyzes suggest that the DNA methylation is dominantly repressedand that it cannot be reversed by a treatment with HDAC inhibitors suchas TSA (Suzuki (2002); Ghoshal (2002), loc. cit.). There are, however,also more recent indications that valproate, a HDAC inhibitor which isalready used in clinics, can lead to the demethylation of DNA (Detich,J. Biol. Chem. 278 (2003), 27586-27592). However, no systematic analyzeshave so far been carried out in this respect.

Clinical Approaches for Reversing Epigenetic Changes

While genetic causes of cancer (such as, e.g., mutations) areirreversible, epigenetic changes contributing their share to thetumorigenesis might possibly be reversible. Thus, the possible treatmentof epitgenetic changes offers new possibilities of therapy for thetreatment of neoplasias (Herman (2003); Momparler (2003); Plass (2002),all loc. cit.; Leone, Clin. Immunol. 109 (2003), 89-102; Claus, Oncogene22 (2003), 6489-6496).

More than 20 years ago, 5-azacytidine has already been developed as ananti-neoplastic medicament and used without the molecular effect of thesubstance being known. Nowadays, it is already used successfully in afurther developed form (Deoxy-5-azacytidine, Decitabine) for thetreatment of myelodysplastic syndromes and secondary leukaemia (Leone(2003), loc. cit.; Lyons, Curr. Opin. Investig. Drugs 4 (2003),1442-1450; Issa, Curr. Opin. Oncol. 15 (2003), 446-451). Due to the invitro observation that HDAC inhibitors can support the reactivation ofmethylated promoters and can act synergistically with demethylatedsubstances, at present pilot studies are carried out throughout theworld, combining the use of both classes of substances (Kalebic (2003);Claus (2003), loc. cit.; Gagnon, Anticancer Drugs 14 (2003), 193-202;Shaker, Leuk. Res. 27 (2003), 437-444).

Detection Methods for the Analyzis of CpG Methylation

The development of detection methods for the analyzis of genomic CpGmethylation has mainly gained importance due to the fact that it hasbeen found that changes in the CpG methylation pattern can be associatedwith diseases such as cancer. At present, there are mainly techniquesknown which are used for the detection of the CpG methylation of knowngene loci (Dahl, Biogerontology 4 (2003), 233-250). Methods allowing ananalyzis of the CpG methylation throughout the genome are lessestablished. In the following, the most common methods for analyzis ofCpG methylation together with their main fields of application aresummarised.

Use of Methylation-Sensitive Restriction Enzymes for the Detection ofCpG Methylation

The methylation status of specific CpG dinucleotides can be determinedusing isoschizomers of bacterial restriction endonucleases which arecharacterised by different sensitivities vis-à-vis 5-methylcytosine.Examples thereof are the enzymes HpaII and MspI—both cut CCGG sequences,HpaII however only if the internal cytosine is not methylated. Someassays are based on the use of methylation-sensitive restrictionenzymes, said assays being used for both the analyzis of individualgenes and analyzis of the CpG methylation throughout the genome. Thefragments of a methylation-sensitive restriction digestion are mostlydetected by means of Southern blot or a genomic PCR of the regionflanking the restriction site (Dahl (2003), loc. cit.). All analyzes ofthe CpG methylation throughout the genome, which have been published upto today, use methylation-sensitive restriction enzymes as a componentof the method. Restriction Landmark Genomic Scanning (RLGS) (Costello,Methods 27 (2002), 144-149), for instance, uses a kind oftwo-dimensional agarose gel electrophorese in which every dimension isdigested with a different methylation-sensitive restriction enzyme toidentify differences in the CpG methylation of two DNA populations.Methylated CpG Island Amplification (MCA) enriches fragments withmethylated SmaI restriction sites and uses LM-PCR for enriching thefragments. Such amplification products have already been successfullyanalyzed by means of Representational Difference Analyzis (RDA) (Smith,Genome Res. 13 (2003), 558-569) or CpG island microarrays (Yan, CancerRes. 6 (2001), 8375-8380).

With regard to the analyzis of the CpG methylation throughout thegenome, all assays that are based on methylation-sensitive restrictionenzymes have disadvantages. In order to carry out the assays in anoptimal way, it has, amongst others, to be guaranteed that allrestriction digestions are completed. The greatest disadvantage is thatthe analyzes merely inform on the methylation status of the cytosineresidues which have been recognised by the methylation-sensitiverestriction enzymes used. The selection of the restriction enzymesautomatically limits the number of detectable sequences—a neutralanalyzis of the CpG methylation is therefore not possible.

Bisulfate Treatment for the Analyzis of the CpG Methylation

The treatment of double-stranded genomic DNA with sodium bisulfate leadsto the deamination of unmethylated cytosine residues into uracilresidues and to the formation of two single strands that are no longercomplementary. During this treatment, 5-methyl cytosine is maintained.The differences in sequence produced in this way form the basis of thedifferentiation between methylated and unmethylated DNA (Frommer, Proc.Natl. Acad. Sci. 889 (1992), 1827-1831). DNA treated with bisulfite canbe used directly in PCR in which uracil residues (previouslyunmethylated cytosine) and thymidine residues are amplified as thymidineand only 5-methylcytosine residues are amplified as cytosine residues.Depending on the application, the primers used for the PCR differentiatebetween methylated and unmethylated sequences or amplify fragmentsindependently of the methylation status. PCR fragments which have beenamplified using non-discriminating primers can, for instance, besequenced directly to determine the share in methylated and unmethylatedCpGs. Further methods make use of the physical differences of such PCRfragments (melting behaviour, single-strand conformation, restrictionsites for restriction enzymes, etc.) for determining the degree ofmethylation (Dahl (2003), loc. cit.). Other methodical approaches thatallow high throughput methylation analyzes utilise the differences insequence for the specific amplification of methylated and unmethylatedsequences by discriminating primers or probes (methylation-specific PCR,methylight PCR) (Dahl (2003), loc. cit.). Bisulfite-induced differencesin the sequence of PCR products can also be found by means ofmethylation-specific oligonucleotide (MSO) microarrays (Shi, J. Cell.Biochem. 88 (2003), 138-143; Adorjan, Nucleic Acid Res. 30 (2002), e21;Gitan, Genome Res. 12 (2002), 158-164).

In contrast to the methylation-sensitive restriction enzymes, the DNAtreated with bisulfite can provide information on the methylation statusof several CpG residues in an amplified genomic fragment. The detectionof CpG methylation by using discriminating primers or probes, however,is limited to the methylation status of single (or few) cytosineresidues. Hence, the information provided by all presently known assaysof the prior art that are suitable for high throughput methylationanalyzis of single gene loci is limited to one or only a few CpGresidues within the gene of interest.

Further Methods for the Detection of CpG Methylation

Antibodies against 5-methyl cytosine recognise CpG methylation indenatured, single-stranded DNA are used mainly for theimmunohistochemical staining of the CpG methylation on the chromosomesof individual, fixed cells.

Already in 1994, the laboratory of A. Bird developed a method forenriching methylated DNA fragments by means of affinity chromatography(Gross, Nat. Genet. 6 (1994), 236-244). A recombinant MECP2 bound to amatrix was used for binding the methylated DNA. Since then thistechnique has been used, improved and combined with further techniquesby other working groups (Shiraishi, Proc. Natl. Acad. Sci. 96 (1999),2913-2918; Brock, Nucleic Acid. Res. 29 (2001), E123). The binding ofstrongly or less strongly methylated genomic sequences to an affinitymatrix depends on the salt concentration which makes it possible toseparate the CpG islands with dense methylation from other sequenceswith a lower methylation density. The disadvantage of this affinitychromatography is the large amount of genomic DNA required (50-100 μg)and the relatively time-consuming procedure.

In view of the foregoing, it is evident that methylation of CpGdinucleotides is an important epigenetic mechanism for controllingtranscriptional activity of a cell. Generally, methylation of CpGdinucleotides correlates with transcriptional inactivity. Yet, duringnormal or degenerated differentiation processes the methylation patternof genloci may change. Accordingly, the reversal of normal methylationpatterns during tumorigenesis can lead to an abnormal repression (oractivation) of genes, for instance, tumor suppressor genes or oncogenes,respectively, and, thus, leading to tumorigenesis. Hence, the detectionof CpG methylated DNA und thus the identification of misregulatedtumor-suppressor genes and/or oncogenes is of outmost clinical interest.As mentioned above, the prior art describes different approaches for thedetection of methylated DNA which, however, suffer from certainshortcomings. For example, the methods of the prior art may not besuitable for high-through put applications or may not reliable detectCpG methylated DNA, particularly if only low amounts of DNA can be madesubject of an analyzis. Thus, there is still a need for further meansand methods for detecting methylated DNA which may overcome theshortcomings and drawbacks of the prior art. Accordingly, the technicalproblem underlying the present invention is to comply with the needsdescribed above.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, in a first aspect the present invention relates to an invitro method for detecting methylated DNA comprising

-   (a) coating a container with a polypeptide capable of binding    methylated DNA;-   (b) contacting said polypeptide with a sample comprising methylated    and/or unmethylated DNA; and-   (c) detecting the binding of said polypeptide to methylated DNA.

As documented in the appended Examples, it was surprisingly found that asingle-tube assay/in vitro method can be safely and reliable employed inthe detection of methylated nucleic acid molecules, in particularCpG-methylated DNA molecules/DNA fragments. The advantages of saidmethod are its fast, sensitive, and reliable detection of preferablymethylated DNA and its ability to analyze target DNA fragments accordingto their methylation degree. In contrast to the prior art, the methodprovided herein does not require bisulfite treatment ormethylation-sensitive restriction and is not limited to detectingsingle/few CpG residues. The information provided may actually be morerelevant than that of other methods of the prior art, since, methylationdensity of a proximal promoter can correlate better with thetranscriptional status of a gene than the methylation status of a-singleCpG residue within the region. Accordingly, a “single-tube” assay isprovided herein, wherein the degree of methylation may be estimatedrelative to a PCR reaction of the (genomic) input DNA.

A preferably homogeneously coated container in accordance with thisinvention, preferably, facilitates that a polypeptide which is capableof binding methylated DNA and which is employed in accordance with themethod described herein has a maximum binding capacity for methylatedDNA. A homogenous coating of the container can be achieved by methodsknown in the art and preferably by the method of the present inventiondescribed herein and/or in the appended Examples. Further, homogeneouscoating can be controlled by methods known in the art, such asCoomassie-Blue staining. The term “container” encompasses any containerwhich is commonly used and/or suitable for scientific and/or diagnosticpurposes. Preferably, said container is composed of the followingmaterials: polystyrene, polyvinyl chloride or polypropylene or the like,more preferably it is composed of polycarbonate. It is also preferredthat polystyrene, polyvinyl chloride, polypropylene or polycarbonate isthermocycler-compatible, i.e. it is preferably heat-stable and/ordurable at different temperatures for different time intervals. It ismoreover preferred that polystyrene, polyvinyl chloride, polypropyleneor polycarbonate are inert to chemical and/or biological agents used inconnection with the method of the present invention.

So far, coatable PCR-tubes have only been used for immuno-polymerasechain reaction (immuno-PCR) (Sano, Science 258 (1992); Adler, BiochemBiophys Res Commun. 308 (2003), 240-250). Immuno-PCR is an antigendetection system, in which a specific DNA molecule is used as themarker. A streptavidin-protein A chimera that possesses tight andspecific binding affinity both for biotin and immunoglobulin G is usedto attach a biotinylated DNA specifically to antigen-monoclonal antibodycomplexes that are immobilized on microtiter plate wells. Then, asegment of the attached DNA is then amplified by PCR. Immuno-PCR iscomparable to traditional ELISA techniques and uses thesandwich-approach with a more sensitive detection system (PCR detectionof the marker DNA). Thus, in contrast to the present invention, whereDNA is the direct subject of detection, Immuno-PCR uses DNA as a means(marker) to detect an antigen.

The term “coating” means that the surface of the container is preferablyentirely coated with a polypeptide which is capable of bindingmethylated DNA, whereby essentially identical amounts of saidpolypeptide are present in each and every area of the surface of saidcontainer. Examples of such binding polypeptides are given herein belowand comprise, inter alia and preferably, a polypeptide belonging to theMethyl-DNA binding protein (MBD) family, and most preferably abifunctional polypeptide comprising the DNA-binding domain of a proteinbelonging to the family of Methyl-CpG binding proteins (MBDs) and an Fcportion of an antibody. Said DNA-binding domain is described hereinbelow. Optionally, said bifunctional polypeptide comprises a polypeptidelinker which is described herein below. Accordingly, said bifunctionalpolypeptide is preferably characterized by the amino acid sequence shownin SEQ ID NO: 2 (FIG. 7) which is encoded by the nucleotide sequenceshown in SEQ ID NO: 1 (FIG. 7)

The term “polypeptide capable of binding methylated DNA” encompasses anypolypeptide which can bind methylated DNA as described herein. Thecapability of binding methylated DNA can be tested by methods known inthe art. The term “polypeptide” when used herein means a peptide, aprotein, or a polypeptide which are used interchangeable and whichencompasses amino acid chains of a given length, wherein the amino acidresidues are linked by covalent peptide bonds. However, peptidomimeticsof such proteins/polypeptides wherein amino acid(s) and/or peptidebond(s) have been replaced by functional analogs are also encompassed bythe invention as well as other than the 20 gene-encoded amino acids,such as selenocysteine. Peptides, oligopeptides and proteins may betermed polypeptides. As mentioned the terms polypeptide and protein areoften used interchangeably herein. The term polypeptide also refers to,and does not exclude, modifications of the polypeptide. Modificationsinclude glycosylation, acetylation, acylation, phosphorylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.,T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York (1983), pgs. 1-12; Seifter, Meth. Enzymol.182 (1990); 626-646, Rattan, Ann. NY Acad. Sci. 663 (1992); 48-62.Preferably, the term “polypeptide” encompasses a polypeptide capable ofbinding methylated DNA. Said term also encompasses a bifunctionalpolypeptide which is capable of binding methylated DNA and itencompasses an anti-methylated DNA antibody. Such polypeptides aredescribed herein and are employed in the method of the present inventionfor detecting methylated DNA.

A “bifunctional polypeptide” means that a polypeptide has, in additionto binding to methylated DNA, preferably to CpG methylated DNA, due toan Fc portion of an antibody which is part of the said bifunctionalpolypeptide, further capabilities. For example, said Fc portionpreferably offers the possibility to conjugate, link or covalentlycouple (a) compound(s) or moieties to said Fc portion. As used herein,the term “covalently coupled” means that the specified compounds ormoieties are either directly covalently bonded to one another, or elseare indirectly covalently joined to one another through an interveningmoiety or moieties, such as a bridge, spacer, or linkage moiety ormoieties. Furthermore, said Fc portion may be used to couple saidbifunctional polypeptide to a container as is described herein. Apreferred bifunctional polypeptide is characterized by the amino acidsequence shown in SEQ ID NO: 2 (FIG. 7). Further preferred bifunctionalpolypeptides are described herein below.

Without being bound by theory, it is believed that the nascentbifunctional polypeptide comprising an methyl-DNA-binding domain and anFc portion of an antibody is folded within a host cell such thatpreferably two polypeptides are joined at their Fc portion in a mannersimilar or, preferably, identical to the constant region of an antibody,resulting in a bifunctional polypeptide as described herein.

It was surprisingly found that said bifunctional polypeptide, preferablybehaving as an antibody-like protein can preferably bind CpG methylatedDNA in an antibody-like manner. That means, the bifunctional polypeptidehas a high affinity and high avidity to its “antigen” which ispreferably methylated DNA that is preferably methylated at CpGdinucleotides. Again, without being bound by theory, the high affinityand avidity of the bifunctional polypeptide employed in the method ofthe present invention for detecting methylated DNA for its “antigen” iscaused by the unique structure of said bifunctional polypeptide. This isbecause, it is assumed that the constant regions form disulfide-bondsbetween immunoglobulin heavy chains of the constant regions of each oftwo polypeptide molecules of said bifunctional polypeptide. Accordingly,preferably an antibody-like structure is formed which closely resemblesthe structure of an antibody.

Moreover, without being bound by theory it is assumed that thisantibody-like structure lends, for example, stability on thebifunctional polypeptide employed in the method of the present inventionfor detecting methylated DNA. This is because, it is described in theart that proteins fused to a constant region of an antibody may confer ahigher stability and half-life of the said protein. In addition, it isbelieved that the antibody-like structure caused by the intermolecularinteraction of the constant regions brings the methyl-DNA-binding domainof one polypeptide of the bifunctional polypeptide used in accordancewith the method of the present invention for detecting methylated DNA inclose proximity to the methyl-DNA-binding domain of another polypeptideof the present invention employed in the method of the presentinvention. This allows bivalent interactions between themethyl-DNA-binding protein(s) and methylated DNA. Accordingly, thebifunctional polypeptide described herein is preferably capable ofbinding to its antigen via two methyl DNA-binding domains which are partof said bifunctional polypeptide. The high affinity binding of thebifunctional polypeptide is, inter alia, also achieved by usingpreferably methyl-DNA-binding domains of proteins instead of thefull-length methyl-DNA-binding protein containing domains for theinteraction with other proteins that may, however, disturb or interferethe unique applicability as described herein which are known tospecifically bind to methylated DNA, preferably, CpG methylated DNA,rather than to unmethylated DNA. The preferred use of themethyl-DNA-binding domain, moreover, is believed to guarantee thatindeed methylated DNA is bound since the detection is direct and notindirect. Most prior art methods can only indirectly detect methylatedDNA by PCR.

These properties award the preferred bifunctional polypeptide to be areliable and easy applicable diagnostic tool which can be employed inthe method of the present invention for detecting methylated DNA. Yet,it can also be employed in methods for, inter alia, isolating, purifyingenriching methylated DNA even if said DNA is only present in very smallamounts, e.g., about more than 10 ng, less than 10 ng, less than 7.5 ng,less than 5 ng, less than 2.5 ng, less than 1000 pg, less than 500 pg,less than 250 pg or about 150 pg as described herein. Accordingly, dueto its antibody-like structure the bifunctional polypeptide describedherein is a robust molecule rendering it to be applicable, for instance,for various applications including multi-step procedures in a singletube assay as is described herein and in the appended Examples.

The term “contacting” includes every technique which causes that apolypeptide which is capable of binding methylated DNA as is describedherein is brought into contact with a sample comprising methylatedand/or unmethylated DNA. Preferably, said sample comprising methylatedand/or unmethylated DNA is transferred preferably by a pipetting stepinto the container which is coated with a polypeptide described hereinwhich is capable of binding methylated DNA.

A further advantage of the method of the present invention for detectingmethylated DNA is that after the container, preferably a PCR tube hasbeen coated, methylated DNA can be bound by a polypeptide which iscapable of binding methylated DNA preferably within 40-50 minutes.Subsequent washing steps which are preferably applied only needpreferably about 5 minutes which renders the herein described method fordetecting methylated DNA a fast and robust method which can be run in ahigh-throughput format that can optionally be automated.

The term “detecting” encompasses any technique which is suitable fordetecting methylated DNA.

In a preferred embodiment the methylated DNA bound by a polypeptidecapable of binding methylated DNA is detected by restriction enzymedigestion, bisulfite sequencing, pyrosequencing or Southern Blot.However, the detection of methylated DNA is not limited to theaforementioned methods but includes all other suitable methods known inthe art for detecting methylated DNA such as RDA, microarrays and thelike. The term “methylated DNA” encompasses preferably methylated DNA,more preferably, CpG methylated DNA including hemi-methylated DNA or DNAmethylated at both strands or single-stranded methylated DNA. The mostimportant example is methylated cytosine that occurs mostly in thecontext of the dinucleotide CpG, but also in the context of CpNpG- andCpNpN-sequences. In principle, however, other naturally occurringnucleotides may also be methylated.

In an alternative, but also preferred embodiment the methylated DNAbound by a polypeptide capable of binding methylated DNA is detected byan amplification technique, preferably PCR, for example, conventional orreal-time PCR including either single or multiplex conventional orreal-time PCR using preferably gene-specific primers.

The term “amplification technique” refers to any method that allows thegeneration of a multitude of identical or essentially identical (i.e. atleast 95% more preferred at least 98%, even more preferred at least 99%and most preferred at least 99.5% such as 99.9% identical) nucleic acidmolecules or parts thereof. Such methods are well established in theart; see Sambrook et al. “Molecular Cloning, A Laboratory Manual”,2^(nd) edition 1989, CSH Press, Cold Spring Harbor. Various PCRtechniques, including real-time PCR are reviewed, for example, by Ding,J. Biochem. Mol. Biol. 37 (2004), 1-10.

As mentioned above, a variety of amplification methods are known in theart, all of which are expected to be useful for detecting methylated DNAbound by a polypeptide described herein which is capable of bindingmethylated DNA in the method of the invention. It is preferred that thedetection in step (c) is effected by PCR. PCR is a powerful techniqueused to amplify DNA millions of fold, by repeated replication of atemplate, in a short period of time. The process utilizes sets ofspecific in vitro synthesized oligonucleotides to prime DNA synthesis.The design of the primers is dependent upon the sequences of the DNAthat is desired to be analyzed. It is known that the length of a primerresults from different parameters (Gillam (1979), Gene 8, 81-97; Innis(1990), PCR Protocols: A guide to methods and applications, AcademicPress, San Diego, USA). Preferably, the primer should only hybridize orbind to a specific region of a target nucleotide sequence. The length ofa primer that statistically hybridizes only to one region of a targetnucleotide sequence can be calculated by the following formula: (¼)^(x)(whereby x is the length of the primer). For example a hepta- oroctanucleotide would be sufficient to bind statistically only once on asequence of 37 kb. However, it is known that a primer exactly matchingto a complementary template strand must be at least 9 base pairs inlength, otherwise no stable-double strand can be generated (Goulian(1973), Biochemistry 12, 2893-2901). It is also envisaged thatcomputer-based algorithms can be used to design primers capable ofamplifying the nucleic acid molecules of the invention. Preferably, theprimers of the invention are at least 10 nucleotides in length, morepreferred at least 12 nucleotides in length, even more preferred atleast 15 nucleotides in length, particularly preferred at least 18nucleotides in length, even more particularly preferred at least 20nucleotides in length and most preferably at least 25 nucleotides inlength. The invention, however, can also be carried out with primerswhich are shorter or longer.

The PCR technique is carried out through many cycles (usually 20-50) ofmelting the template at high temperature, allowing the primers to annealto complimentary sequences within the template and then replicating thetemplate with DNA polymerase. The process has been automated with theuse of thermostable DNA polymerases isolated from bacteria that grow inthermal vents in the ocean or hot springs. During the first round ofreplication a single copy of DNA is converted to two copies and so onresulting in an exponential increase in the number of copies of thesequences targeted by the primers. After just 20 cycles a single copy ofDNA is amplified over 2,000,000 fold.

In a preferred embodiment, the aforementioned method further comprisesstep (d) analyzing the DNA bound by a polypeptide capable of binding tomethylated DNA. The analyzis is preferably done by sequencing. Saidsequencing is preferably performed by methods known in the art, forexample, automated didesoxy-sequencing using fluorescent didesoxynucleotides according to the method of Sanger (Proc. Natl. Acad. Sci. 74(1977), 5463-5467). For automated sequencing the DNA to be sequenced isprepared according to methods known in the art and preferably accordingto the instructions of the kit used for preparing said DNA forsequencing.

Before the present invention is described in detail, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, bacteria, vectors, and reagents etc. describedherein as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Throughoutthis specification and the claims which follow, unless the contextrequires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the”, include plural referents unless thecontext clearly indicates otherwise. Thus, for example, reference to “areagent” includes one or more of such different reagents, and referenceto “the method” includes reference to equivalent steps and methods knownto those of ordinary skill in the art that could be modified orsubstituted for the methods described herein.

CpG islands frequently contain gene promoters and transcription startsites and are usually unmethylated in normal cells. Methylation ofCpG-islands is associated with transcriptional repression. In cancer,the methylation of CpG-island promoters leads to the abnormal silencingof tumor-suppressor genes, contributing to the pathogenesis of thedisease. As mentioned above, the prior art describes differentapproaches for the detection of methylated candidate genes which,however, suffer from certain shortcomings. For example, high throughputmethods of the prior art may be limited to the detection of single/fewCpG residues or may not reliable detect CpG methylated DNA, particularlyif only low amounts of DNA can be made subject of an analyzis. To allowa rapid and sensitive detection of the degree of CpG-methylation ofcandidate genes, the present invention provides means and methods thatallow the detection of CpG-methylation, without applying, for example,methylation-sensitive restriction endonucleases or bisulfite-treatment.

In addition to the surprising finding mentioned herein above as regardsthe method of the present invention for detecting methylated DNA, it wasfurther surprisingly found that binding of methylated DNA and/orfragments thereof to the relatively small surface of containers,preferably PCR-tubes is sufficient to detect preferably a single genelocus within a complex mixture of methylated and/or methylated DNAand/or fragments thereof. Accordingly, it was found that preferably aone-tube assay for detecting methylated DNA termed methyl-binding(MB)-PCR is a reliable and easy applicable diagnostic tool for, interalia, isolating, purifying, enriching and/or preferably detectingmethylated DNA even if said DNA is only present in very small amounts,e.g., about more than 10 ng, less than 10 ng, less than 7.5 ng, lessthan 5 ng, less than 2.5 ng, less than 1000 pg, less than 500 pg, lessthan 250 pg or about 150 pg as described herein. Using the methods andkits described herein, it is possible to generate methylation profilesof single or multiple gene loci in for example, human cancer in largenumbers of samples.

Briefly, a preferred embodiment of the method of the present inventionfor detecting methylated DNA is MB-PCR which may work as follows:

A protein with preferably high affinity for methylated DNA, inparticular for CpG-methylated DNA, is coated onto the walls ofpreferably a PCR-cycler compatible reaction container, preferably a tubeand used to selectively capture methylated DNA and/or DNA-fragments frompreferably a genomic DNA mixture. The retention of a specific DNA and/orDNA-fragment (e.g. a CpG island promoter of a specific gene) can bedetected in the same container using PCR (either standard PCR orrealtime PCR, single or multiplex). The degree of methylation may beestimated relative to a PCR reaction of the genomic input DNA. Thus, thepresent invention provides a quick, simple, reliable, robust andextremely sensitive technique allowing the detection of methylated DNA,in particular in tumorous tissue or tumor cells from limited samples.

The preferred diagnostic application employing a polypeptide capable ofbinding methylated DNA is shown in FIG. 1A. FIG. 1B shows the preferreddiagnostic application by employing a bifunctional polypeptide which iscapable of binding CpG-methylated DNA as described herein. Briefly, in afirst step preferably a methyl-CpG-binding polypeptide is preferablyadded into a coatable PCR-vessel, for example, TopYield Strips fromNunc. In doing so, the polypeptide is preferably coated onto the innersurface of said vessel by techniques known in the art and describedherein. In a next step, blocking reagents, e.g., about 5% milk powderare added into the coated PCR vessel. In a further step, preferablyDNA-fragments of interest (for example, methylated and/or unmethylatedDNA-fragments (the term “CpG-methylation low” used in FIGS. 1A and Bcomprises and particularly refers to unmethylated DNA)) are added intothe coated and blocked PCR vessel. It is believed that themethyl-CpG-binding polypeptide binds specifically to methylated DNA, ifpresent. In a following step, the coated and blocked PCR vesselcontaining preferably DNA-fragments is incubated and then washed toremove unbound DNA-fragments. Afterwards, a PCR mix including preferablygene-specific primers or, but also preferred, at least two, three, four,five, six, seven etc. pairs of primers for, e.g., multiplex PCR for thegene or genlocus or genloci of interest which is/are suspected to bemethylated or unmethylated is added to run preferably, a real time PCRor conventional PCR followed by gel electrophoresis to separateamplification products. Optionally, a control reaction can be performedas is shown in FIG. 1A or 1B as “P-reaction” which is describedhereinbelow.

A preferred detailed protocol for MB-PCR is as follows:

Preferably, the PCR tubes are prepared using heat stable TopYield™Strips (Nunc Cat. No. 248909). Preferably, 50 μl of the a polypeptidedescribed herein, preferably a methyl-CpG-binding polypeptide (dilutedat 15 μg/ml in 10 mM Tris/HCl pH 7.5) are added to each well andincubated overnight at 4° C. Preferably, wells are washed three timeswith 200 μl TBS (20 mM Tris, pH 7.4 containing 170 mM NaCl) and blockedpreferably for 3-4 h at RT with 100 μl Blocking Solution (10 mM Tris, pH7.5 containing 170 mM NaCl, 5% skim milk powder, 5 mM EDTA and 1 μg/mlof each poly d(I/C), poly d(A/T) and poly d(CG)). Preferably, tubes arethen washed three times with 200 μl TBST (TBS containing 0.05%Tween-20).

Preferably, 50 μl Binding Buffer (20 mM Tris, pH 7.5 containing 400 mMNaCl, 2 mM MgCl₂, 0.5 mM EDTA, and 0.05% Tween-20) are added to eachwell and preferably 2 μl of digested DNA, preferably genomic DNAdigested with MseI in an amount of preferably 5 ng/μl is added to everysecond well (M-reaction).

Genomic DNA is preferably prepared by using a kit known in the art, forexample, using Blood and Cell Culture Midi Kit (Qiagen). The quality ofthe genomic DNA-preparation is preferably controlled by agarose gelelectrophoresis and DNA concentration was preferably determined by UVspectrophotometry. Quantitation of DNA is preferably done by usingPicoGreen dsDNA Quantitation Reagent (Molecular Probes).

The wells containing a polypeptide described herein and DNA, preferablyDNA-fragments (generated by enzymatic digestion or mechanicallyfragmented) are incubated on a shaker at preferably RT for preferably40-50 min. Preferably, tubes were washed two times with 200 μl BindingBuffer and once with 10 mM Tris/HCl pH 8.0.

Next, PCR is preferably carried out directly in the TopYield™ Strips.Preferably, the PCR-Mix (50 μl/well), preferably PCR Master Mix(Promega), contains preferably 10 pmol of each gene-specific primer(synthesized by Metabion). Primer sequences and cycling parameters forspecific genes of interest are given in the Example. Of course, anyother suitable gene specific or genlocus specific or genloci specificprimers can be designed by the person skilled in the art. Moreover, theskilled artisan can readily determine and/or test the PCR parametersmost suitable for the primer(s) and gene(s), genlocus/genloci ofinterest. After adding the PCR-mix, preferably 1 μl Mse I-digested DNA(preferably in an amount of 5 ng/μl) is added to every second otherwell, that was not previously incubated with DNA-fragments (P-reaction).Preferably, PCR-products are analyzed using agarose gel electrophoresisand the ethidium bromide stained gel was scanned using, for example, aTyphoon 9200 Imager (Amersham/Pharmacia).

Optionally, a control reaction can be performed as is shown in FIG. 1Aor 1B as “P-reaction” which is described hereinbelow.

Accordingly, it is envisaged that the method of the present invention isuseful for the detection of methylated DNA, preferably CpG-methylatedDNA in a sample as described herein below which may include (a) singlecell(s). It is also envisaged to be useful for whole cells. “Whole cell”means the genomic context of a whole single cell.

In the following, preferred polypeptides capable of binding methylatedDNA are described. Accordingly, a polypeptide used in the methods of thepresent invention for detecting methylated DNA is preferably selectedfrom the group consisting of

-   (a) a polypeptide belonging to the Methyl-DNA binding protein (MBD)    family;-   (b) a fragment of the polypeptide of (a), wherein said fragment is    capable of binding methylated DNA;-   (c) a variant of the polypeptide of (a) or the fragment of (b),    wherein in said variant one or more amino acid residues are    substituted compared to the polypeptide of (a) or the fragment of    (b), and wherein said variant is capable of binding methylated DNA;-   (d) a polypeptide which is an anti-methylated DNA antibody or    fragment thereof; and-   (e) a polypeptide which is at least 70% identical to a polypeptide    of any one of (a) to (c) and which is capable of binding methylated    DNA.

Of course, it is envisaged that the herein described polypeptides whichare capable of detecting methylated DNA are encoded by a nucleic acidmolecule. The term “nucleic acid molecule” when used herein encompassesany nucleic acid molecule having a nucleotide sequence of basescomprising purine- and pyrimidine bases which are comprised by saidnucleic acid molecule, whereby said bases represent the primarystructure of a nucleic acid molecule. Nucleic acid sequences includeDNA, cDNA, genomic DNA, RNA, synthetic forms, for example, PNA, andmixed polymers, both sense and antisense strands, or may containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those skilled in the art. The polynucleotide of thepresent invention encoding a polypeptide which is capable of bindingmethylated DNA and which is employed in the method of the presentinvention is preferably composed of any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, the polynucleotide can be composed of single-and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the polynucleotide can be composed of triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The polynucleotide may alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, the term “nucleic acidmolecules” embraces chemically, enzymatically, or metabolically modifiedforms.

In an alternative, but also preferred embodiment, a bifunctionalpolypeptide (i.e. the MBD protein to be employed in the methods and kitsprovided herein) capable of binding methylated DNA which is employed inthe method of the present invention is encoded by a nucleic acidmolecule comprising a nucleotide sequence of the present inventiondescribed hereinabove is selected from the group consisting of:

-   (a) a nucleic acid sequence having the nucleotide sequence shown in    SEQ ID NO: 1 (FIG. 7);-   (b) a nucleic acid sequence having a nucleotide sequence encoding a    polypeptide having the amino acid sequence shown in SEQ ID: NO 2    (FIG. 7);-   (c) a nucleic acid sequence having a nucleotide sequence encoding a    fragment of a polypeptide having the amino acid sequence shown in    SEQ ID: NO 2 (FIG. 7), wherein said fragment comprises at least    amino acids 130 to 361 of said polypeptide and which is capable of    binding methylated DNA;-   (d) a nucleic acid sequence having a nucleotide sequence encoding a    variant of a polypeptide encoded by a polynucleotide of any one    of (a) to (c), wherein in said variant one or more amino acid    residues are substituted compared to said polypeptide, and said    variant is capable of binding methylated DNA;-   (e) a nucleic acid sequence having a nucleotide sequence which    hybridizes with a nucleic acid sequence of any one of (a) to (d) and    which is at least 65% identical to the nucleotide sequence of the    nucleic acid molecule of (a) and which encodes a polypeptide being    capable of binding methylated DNA;-   (f) a nucleic acid molecule encoding a polypeptide which is at least    65% identified to a polypeptide encoded by a nucleic acid molecule    of (b) and which is capable of binding methylated DNA; and-   (g) a nucleic acid sequence having a nucleotide sequence being    degenerate to the nucleotide sequence of the polynucleotide of any    one of (a) to (f);    or the complementary strand of such a polynucleotide.

The above embodiment relates, accordingly, e.g. to the use of a “MBD-Fc”molecule in the kits and methods provided herein.

As described above, a fragment of a bifunctional polypeptide employed inthe method of the present invention for detecting methylated DNA whichhas the amino acid sequence shown in SEQ ID: NO 2 (FIG. 7) comprises atleast amino acids 130 to 361 of the amino acid sequence shown in SEQ ID:NO 2 (FIG. 7). That means that said fragment may comprise in addition toamino acids 130 to 361 which represent the Fc portion, one or more aminoacids such that said fragment is capable of binding methylated DNA,preferably, CpG methylated DNA, rather than unmethylated DNA.Accordingly, it is envisaged that said fragment comprises morepreferably, at least amino acids 116 to 361 of the amino acid sequenceshown in SEQ ID: NO 2 (FIG. 7). Even more preferably, said fragment maycomprise at least amino acids 29 to 115 and 130 to 361 of the amino acidsequence shown in SEQ ID: NO 2 (FIG. 7). In a most preferred embodiment,said fragment may comprise at least amino acids 29 to 361. It isgenerally preferred that the fragments of the a polypeptide describedherein are able to bind to methylated DNA, preferably to CpG methylatedDNA, rather than unmethylated DNA. This ability can be tested by methodsknown in the art or preferably by those methods described in theappended Examples.

The present invention preferably also relates to methods, whereinnucleic acid sequences which hybridize to the nucleic acid sequenceencoding a polypeptide which is capable of binding methylated DNA areemployed. Said hybridizing nucleic acids encode a polypeptide which iscapable of binding methylated DNA: Moreover, in the methods of thepresent invention, nucleic acids are employed which hybridize to thesequences shown in SEQ ID NO: 1 or fragments or variants thereof asdescribed herein (FIG. 7) and which are at least 65% identical to thenucleic acid sequence shown in SEQ ID NO: 1 (FIG. 7) and whichpreferably encode a bifunctional polypeptide being capable of bindingmethylated DNA, preferably CpG methylated DNA, rather than unmethylatedDNA, wherein said polypeptide is employed in the method of the presentinvention for detecting methylated DNA. Furthermore, the presentinvention preferably relates to methods in which nucleic acid sequencesencoding a polypeptide are employed which are at least 65%, morepreferably 70%, 75%, 80%, 85%, 90%, more preferably 99% identical to apolypeptide as described herein which is capable of binding methylatedDNA. It is also preferably envisaged that in the methods of the presentinvention polypeptides are employed which are at least 65%, morepreferably 70%, 75%, 80%, 85%, 90%, more preferably 99% identical to thepolypeptide shown in SEQ ID NO:2. The term “hybridizes” as used inaccordance with the present invention preferably relates tohybridizations under stringent conditions. The term “hybridizingsequences” preferably refers to sequences which display a sequenceidentity of at least 65%, even more preferably at least 70%,particularly preferred at least 80%, more particularly preferred atleast 90%, even more particularly preferred at least 95% and mostpreferably at least 97, 98% or 99% identity with a nucleic acid sequenceas described above encoding a polypeptide which is capable of bindingmethylated DNA or a bifunctional polypeptide which is able to bind tomethylated DNA, preferably CpG methylated DNA, rather than unmethylatedDNA, wherein said polypeptide capable of binding methylated DNA or saidbifunctional polypeptide is employed in the method of the presentinvention for detecting methylated DNA.

Said hybridization conditions may be established according toconventional protocols described, for example, in Sambrook, Russell“Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory,N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), or Higginsand Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRLPress Oxford, Washington D.C., (1985). The setting of conditions is wellwithin the skill of the artisan and can be determined according toprotocols described in the art. Thus, the detection of only specificallyhybridizing sequences will usually require stringent hybridization andwashing conditions such as 0.1×SSC, 0.1% SDS at 65° C. Non-stringenthybridization conditions for the detection of homologous or not exactlycomplementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is wellknown, the length of the probe and the composition of the nucleic acidto be determined constitute further parameters of the hybridizationconditions. Note that variations in the above conditions may beaccomplished through the inclusion and/or substitution of alternateblocking reagents used to suppress background in hybridizationexperiments. Typical blocking reagents include Denhardt's reagent,BLOTTO, heparin, denatured salmon sperm DNA, and commercially availableproprietary formulations. The inclusion of specific blocking reagentsmay require modification of the hybridization conditions describedabove, due to problems with compatibility. Hybridizing nucleic acidmolecules also comprise fragments of the above described molecules. Suchfragments may represent nucleic acid sequences as described herein.Furthermore, nucleic acid molecules which hybridize with any of theaforementioned nucleic acid molecules also include complementaryfragments, derivatives and allelic variants of these molecules.Additionally, a hybridization complex refers to a complex between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary G and C bases and between complementary A and Tbases; these hydrogen bonds may be further stabilized by base stackinginteractions. The two complementary nucleic acid sequences hydrogen bondin an antiparallel configuration. A hybridization complex may be formedin solution (e.g., Cot or Rot analyzis) or between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., membranes, filters, chips, pins orglass slides to which, e.g., cells have been fixed). The termscomplementary or complementarity refer to the natural binding ofpolynucleotides under permissive salt and temperature conditions bybase-pairing. For example, the sequence “A-G-T” binds to thecomplementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, in which only some of thenucleic acids bind, or it may be complete when total complementarityexists between single-stranded molecules. The degree of complementartitybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, which depend uponbinding between nucleic acids strands.

Moreover, the present invention also relates to methods employed nucleicacid molecules the sequence of which is degenerate in comparison withthe sequence of an above-described nucleic acid molecules, wherein suchdegenerate nucleic acid molecules encode a polypeptide which is capableof binding methylated DNA or which encode a bifunctional polypeptide asdescribed herein and which is employed in the method of the presentinvention for detecting methylated DNA. When used in accordance with thepresent invention the term “being degenerate as a result of the geneticcode” means that due to the redundancy of the genetic code differentnucleotide sequences code for the same amino acid.

Of course, the present invention also envisages the complementary strandto the aforementioned and below mentioned nucleic acid molecules if theymay be in a single-stranded form.

Preferably, the nucleic acid molecule encoding a polypeptide which iscapable of binding methylated DNA or a bifunctional polypeptide capableof binding methylated DNA and which is/are employed in the method of thepresent invention may be any type of nucleic acid, e.g. DNA, genomicDNA, cDNA, RNA or PNA (peptide nucleic acid).

For the purposes of the present invention, a peptide nucleic acid (PNA)is a polyamide type of DNA analog and the monomeric units for adenine,guanine, thymine and cytosine are available commercially (PerceptiveBiosystems). Certain components of DNA, such as phosphorus, phosphorusoxides, or deoxyribose derivatives, are not present in PNAs. Asdisclosed by Nielsen et al., Science 254:1497 (1991); and Egholm et al.,Nature 365:666 (1993), PNAs bind specifically and tightly tocomplementary DNA strands and are not degraded by nucleases. In fact,PNA binds more strongly to DNA than DNA itself does. This is probablybecause there is no electrostatic repulsion between the two strands, andalso the polyamide backbone is more flexible. Because of this, PNA/DNAduplexes bind under a wider range of stringency conditions than DNA/DNAduplexes, making it easier to perform multiplex hybridization. Smallerprobes can be used than with DNA due to the strong binding. In addition,it is more likely that single base mismatches can be determined withPNA/DNA hybridization because a single mismatch in a PNA/DNA 15-merlowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for theDNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA meansthat hybridization can be done at low ionic strengths and reducepossible interference by salt during the analyzis.

The DNA may, for example, be genomic DNA or cDNA. The RNA may be, e.g.,mRNA. The nucleic acid molecule may be natural, synthetic orsemisynthetic or it may be a derivative, such as peptide nucleic acid(Nielsen, Science 254 (1991), 1497-1500) or phosphorothioates.Furthermore, the nucleic acid molecule may be a recombinantly producedchimeric nucleic acid molecule comprising any of the aforementionednucleic acid molecules either alone or in combination.

The nucleic acid molecule encoding a polypeptide described herein whichis employed in the method of the present invention for detectingmethylated DNA is envisaged to be contained in a vector (e.g. a plasmid,cosmid, virus, bacteriophage) which may be transformed into a host cell(a prokaryotic or eukaryotic cell) so as to, inter alia, produce apolypeptide of the present invention which is employed in the method ofthe present invention. A polypeptide of the invention which is employedin the method of the present invention may be produced bymicrobiological methods or by transgenic mammals. It is also envisagedthat a polypeptide of the invention is recovered from transgenic plants.Alternatively, a polypeptide of the invention may be producedsynthetically or semi-synthetically.

For example, chemical synthesis, such as the solid phase proceduredescribed by Houghton Proc. Natl. Acad. Sci. USA (82) (1985), 5131-5135,can be used. Another method is in vitro translation of mRNA. A preferredmethod involves the recombinant production of protein in host cells asdescribed above. For example, nucleotide acid sequences comprising allor a portion of any one of the nucleotide sequences according to theinvention can be synthesized by PCR, inserted into an expression vector,and a host cell transformed with the expression vector. Thereafter, thehost cell is cultured to produce the desired polypeptide, which isisolated and purified. Protein isolation and purification can beachieved by any one of several known techniques; for example and withoutlimitation, ion exchange chromatography, gel filtration chromatographyand affinity chromatography, high pressure liquid chromatography (HPLC),reversed phase HPLC, preparative disc gel electrophoresis. In addition,cell-free translation systems can be used to produce a polypeptides ofthe present invention. Suitable cell-free expression systems for use inaccordance with the present invention include rabbit reticulocytelysate, wheat germ extract, canine pancreatic microsomal membranes, E.coli S30 extract, and coupled transcription/translation systems such asthe TNT-system (Promega). These systems allow the expression ofrecombinant polypeptides or peptides upon the addition of cloningvectors, DNA fragments, or RNA sequences containing coding regions andappropriate promoter elements. As mentioned supra, proteinisolation/purification techniques may require modification of theproteins of the present invention using conventional methods. Forexample, a histidine tag can be added to the protein to allowpurification on a nickel column. Other modifications may cause higher orlower activity, permit higher levels of protein production, or simplifypurification of the protein. After production of a polypeptide which isemployed in the method of the present invention it may be modified bypegylation, derivatization and the like.

The term “polypeptide belonging to the Methyl-DNA binding protein (MBD)”encompasses a polypeptide which has preferably the structural and/orfunctional characteristics of the methyl-DNA-binding domain (MBD) of aprotein of the MBD family which comprises the proteins MeCP2, MBD1,MBD2, MBD3 and MBD4. Said term also encompasses polypeptides with thecapability of binding methylated DNA, including, inter alia, antibodiesraised against methylated DNA. Preferably, said antibody is ananti-5-methylcysteine antibody or fragment thereof. Preferably, saidfragment is a Fab, F(ab′)₂, Fv or scFv fragment. The methyl-DNA-bindingactivity can be tested by methods known in the art. It is preferred thata polypeptide described herein binds methylated DNA either as a monomeror dimer or multivalent molecule as described elsewhere herein. It ispreferably capable of binding to highly methylated DNA or low methylatedDNA. Preferably, it can bind single methylated CpG pairs. MeCP2, MBD1,MBD2, MBD3 and MBD4 constitute a family of vertebrate proteins thatshare the methyl-CpG-binding domain. The MBD protein family comprisestwo subgroups based upon sequences of the known MBDs. Themethyl-DNA-binding domain of MBD4 is most similar to that of MeCP2 inprimary sequence, while the methyl-DNA-binding domain of MBD1, MBD2 andMBD3 are more similar to each other than to those of either MBD4 orMeCP2. However, the methyl-DNA-binding domains within each proteinappear to be related evolutionarily based on the presence of an intronlocated at a conserved position within all five genes of MeCP2, MBD1,MBD2, MBD3 and MBD4. Yet, the sequence similarity between the members ofthe MBD family is largely limited to their methyl-DNA-binding domain,although MBD2 and MBD3 are similar and share about 70% of overallidentity over most of their length. The greatest divergence occurs atthe C-terminus, where MBD3 has 12 consecutive glutamic acid residues.

A protein belonging to the MBD family or fragment thereof, preferably amethyl-DNA-binding domain, useful in accordance with the methods of thepresent invention can, for example, be identified by using sequencecomparisons and/or alignments by employing means and methods known inthe art, preferably those described herein and comparing and/or aligning(a) known MBD(s) to/with a sequence suspected to be an MBD.

For example, when a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit (for instance,if a position in each of the two DNA molecules is occupied by adenine,or a position in each of two polypeptides is occupied by a lysine), thenthe respective molecules are identical at that position. The percentageidentity between two sequences is a function of the number of matchingor identical positions shared by the two sequences divided by the numberof positions compared×100. For instance, if 6 of 10 of the positions intwo sequences are matched or are identical, then the two sequences are60% identical. By way of example, the DNA sequences CTGACT and CAGGTTshare 50% homology (3 of the 6 total positions are matched). Generally,a comparison is made when two sequences are aligned to give maximumhomology and/or identity. Such alignment can be provided using, forinstance, the method of Needleman, J. Mol. Biol. 48 (1970): 443-453,implemented conveniently by computer programs such as the Align program(DNAstar, Inc.). Homologous sequences share identical or similar aminoacid residues, where similar residues are conservative substitutionsfor, or “allowed point mutations” of, corresponding amino acid residuesin an aligned reference sequence. In this regard, a “conservativesubstitution” of a residue in a reference sequence are thosesubstitutions that are physically or functionally similar to thecorresponding reference residues, e.g., that have a similar size, shape,electric charge, chemical properties, including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran “accepted point mutation” in Dayhoff et al., 5: Atlas of ProteinSequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed.Res. Foundation, Washington, D.C. (1978).

Preferably, a fragment of a polypeptide described herein and employed inthe method of the present invention which is capable of bindingmethylated DNA, preferably, a methyl-DNA-binding domain or fragmentthereof of a polypeptide employed in the method of the presentinvention, has preferably the structural and/or functionalcharacteristics of a protein belonging to the MBD-family as describedherein. Preferably, a fragment of a methyl-DNA-binding protein describedherein is able to bind methylated DNA, preferably CpG methylated DNA.

The methyl-DNA-binding domain or fragment thereof of a polypeptide ofthe present invention which is employed in the method of the presentinvention is preferably of insect origin, nematode origin, fish origin,amphibian origin, more preferably of vertebrate origin, even morepreferably of mammal origin, most preferably of mouse and particularlypreferred of human origin.

Preferably, the methyl-DNA-binding domain or fragment thereof of apolypeptide of the present invention which is employed in the method ofthe present invention possesses a unique alpha-helix/beta-strandsandwich structure with characteristic loops as is shown in FIG. 1 ofBallester and Wolffe, Eur. J. Biochem. 268 (2001), 1-6 and is able tobind methylated DNA.

More preferably, the protein belonging to the MBD family or fragmentthereof of a polypeptide of the present invention which is employed inthe method of the present invention comprises at least 50, morepreferably at least 60, even more preferably at least 70 or at least 80amino acid residues of the MBDs shown in FIG. 1 of Ballester and Wolffe(2001), loc. cit. and is able to bind methylated DNA.

Even more preferably, the methyl-DNA-binding domain or fragment orvariant thereof of a polypeptide of the present invention employed inthe method of the present invention shares preferably 50%, 60%, 70%, 80%or 90%, more preferably 95% or 97%, even more preferably 98% and mostpreferably 99% identity on amino acid level to the MBDs shown in FIG. 1of Ballester and Wolffe (2001), loc. cit. and is able to bind methylatedDNA. Means and methods for determining the identity of sequences, forexample, amino acid sequences is described elsewhere herein.

In accordance with the present invention, the term “identical” or“percent identity” in the context of two or more nucleic acid or aminoacid sequences, refers to two or more sequences or subsequences that arethe same, or that have a specified percentage of amino acid residues ornucleotides that are the same (e.g., at least 65% identity, preferably,at least 70-95% identity, more preferably at least 95%, 96%, 97%, 98% or99% identity), when compared and aligned for maximum correspondence overa window of comparison, or over a designated region as measured using asequence comparison algorithm as known in the art, or by manualalignment and visual inspection. Sequences having, for example, 65% to95% or greater sequence identity are considered to be substantiallyidentical. Such a definition also applies to the complement of a testsequence. Preferably the described identity exists over a region that isat least about 232 amino acids or 696 nucleotides in length. Thosehaving skill in the art will know how to determine percent identitybetween/among sequences using, for example, algorithms such as thosebased on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994),4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), asknown in the art.

Although the FASTDB algorithm typically does not consider internalnon-matching deletions or additions in sequences, i.e., gaps, in itscalculation, this can be corrected manually to avoid an overestimationof the % identity. CLUSTALW, however, does take sequence gaps intoaccount in its identity calculations. Also available to those havingskill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl.Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acidsequences uses as defaults a word length (W) of 11, an expectation (E)of 10, M=5, N=4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3,and an expectation (E) of 10. The BLOSUM62 scoring matrix (HenikoffProc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

For example, BLAST2.0, which stands for Basic Local Alignment SearchTool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol.Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410),can be used to search for local sequence alignments. BLAST producesalignments of both nucleotide and amino acid sequences to determinesequence similarity. Because of the local nature of the alignments,BLAST is especially useful in determining exact matches or inidentifying similar sequences. The fundamental unit of BLAST algorithmoutput is the High-scoring Segment Pair (HSP). An HSP consists of twosequence fragments of arbitrary but equal lengths whose alignment islocally maximal and for which the alignment score meets or exceeds athreshold or cutoff score set by the user. The BLAST approach is to lookfor HSPs between a query sequence and a database sequence, to evaluatethe statistical significance of any matches found, and to report onlythose matches which satisfy the user-selected threshold of significance.The parameter E establishes the statistically significant threshold forreporting database sequence matches. E is interpreted as the upper boundof the expected frequency of chance occurrence of an HSP (or set ofHSPs) within the context of the entire database search. Any databasesequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.;Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used tosearch for identical or related molecules in nucleotide databases suchas GenBank or EMBL. This analyzis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score which is defined as:

$\frac{\% \mspace{14mu} {sequence}\mspace{14mu} {identity}\; \times \; \% \mspace{14mu} {maximum}\mspace{14mu} {BLAST}\mspace{14mu} {{sco}{re}}}{100}$

and it takes into account both the degree of similarity between twosequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Similar molecules are usually identified byselecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

Most preferably, the methyl-DNA-binding domain or fragment or variantthereof of a polypeptide of the present invention employed in the methodof the present invention comprises the methyl-DNA-binding domain of theMBD proteins shown in FIG. 1 of Ballester and Wolffe (2001), loc. cit.or the methyl-DNA-binding domain of the MBD proteins described inHendrich and Tweedy, Trends Genet. 19 (2003), 269-77 and is able to bindmethylated DNA.

In a particular preferred embodiment of the invention, themethyl-DNA-binding domain of a polypeptide employed in the method of thepresent invention is that of human MBD2. In a more particular preferredembodiment, the methyl-DNA-binding domain is that of human MBD2comprising amino acids 144 to 230 of the amino acid sequence havingGenbank accession number NM_(—)003927. In a most particular preferredembodiment, the methyl-DNA-binding domain of a polypeptide employed inthe method of the present invention comprises the amino acid sequencefrom position 29 to 115 of the amino acid sequence shown in SEQ ID NO:2(FIG. 3).

A “variant” of a polypeptide of the present invention which is capableof binding methylated DNA and which is employed in the method of thepresent invention encompasses a polypeptide wherein one or more aminoacid residues are substituted, preferably conservatively substitutedcompared to said polypeptide and wherein said variant is preferably ableto bind to methylated DNA, preferably CpG methylated DNA. Such variantsinclude deletions, insertions, inversions, repeats, and substitutionsselected according to general rules known in the art so as have noeffect on the activity of a polypeptide of the present invention. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, Science 247: (1990) 1306-1310,wherein the authors indicate that there are two main strategies forstudying the tolerance of an amino acid sequence to change. The firststrategy exploits the tolerance of amino acid substitutions by naturalselection during the process of evolution. By comparing amino acidsequences in different species, conserved amino acids can be identified,These conserved amino acids are likely important for protein function.In contrast, the amino acid positions where substitutions have beentolerated by natural selection indicates that these positions are notcritical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein. The second strategy uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene toidentify regions critical for protein function. For example, sitedirected mutagenesis or alanine-scanning mutagenesis (introduction ofsingle alanine mutations at every residue in the molecule) can be used.(Cunningham and Wells, Science 244: (1989) 1081-1085.) The resultingmutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved.

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thefunctions performed by a polypeptide as described herein which isemployed in the method of the present invention. Similarity isdetermined by conserved amino acid substitution. Such substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics (e.g., chemical properties).According to Cunningham et al. above, such conservative substitutionsare likely to be phenotypically silent. Additional guidance concerningwhich amino acid changes are likely to be phenotypically silent arefound in Bowie, Science 247: (1990) 1306-1310.

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in the Table below.

TABLE IV For Amino Aeid Code Replace with any of: Alanine A D-Ala, Gly,beta-Ala, L-Cys, D-C_(y)s Arginine R D-Arg, Lys, D-Lys, homo-Arg,D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp,D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-As Glutamic Acid ED-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro,D-Pro, β-Ala, Acp Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine MD-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine FD-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylicacid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(0),L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Infoliuaties and Genome Projects, Smith, DM., ed., Academic Press, NewYork, 1993; Informafies Computer Analyzis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analyzis in Molecular Biology, von Heinje, G., AcademiePress, 1987; and Sequence Analyzis Primer, Gribskov, M. and Devereux,eds., M Stockton Press, New York, 1991.

As mentioned herein above, a polypeptide to be used for bindingmethylated DNA also encompasses preferably an anti-methylated DNAantibody which is preferably an anti-5-methylcytosine antibody or a Fab,F(ab′)₂, Fv or scFv fragment thereof. Preferably, saidanti-5-methylcytosine antibody specifically binds to methylated DNA,preferably CpG-methylated DNA. The term “specifically” in this contextmeans that said antibody reacts with CpG-methylated DNA, but not withunmethylated DNA and/or DNA methylated at other nucleotides thancytosine and/or DNA methylated at other positions than the C5 atom ofcytosine.

Whether the antibody specifically reacts as defined herein above caneasily be tested, inter alia, by comparing the binding reaction of saidantibody with CpG-methylated DNA and with unmethylated DNA and/or DNAmethylated at other nucleotides than cytosine and/or DNA methylated atother positions than the C5 atom of cytosine.

The antibody of the present invention can be, for example, polyclonal ormonoclonal. The term “antibody” also comprises derivatives or fragmentsthereof which still retain the binding specificity such as a Fab,F(ab′)₂, Fv or scFv fragment. Techniques for the production ofantibodies are well known in the art and described, e.g. in Harlow andLane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor,1988. The present invention furthermore includes chimeric, single chainand humanized antibodies, as well as antibody fragments as mentionedabove; see also, for example, Harlow and Lane, loc. cit. Variousprocedures are known in the art and may be used for the production ofsuch antibodies and/or fragments. Thus, the (antibody) derivatives canbe produced by peptidomimetics. Further, techniques described for theproduction of single chain antibodies (see, inter alia, U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptide(s) of this invention. Also, transgenic animals may be usedto express humanized antibodies to polypeptides of this invention. Mostpreferably, the anti-methylated DNA antibody of this invention is amonoclonal antibody. For the preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples for such techniques include the hybridomatechnique (Köhler and Milstein Nature 256 (1975), 495-497), the triomatechnique, the human B-cell hybridoma technique (Kozbor, ImmunologyToday 4 (1983), 72) and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing theproduction of single chain antibodies (e.g., U.S. Pat. No. 4,946,778)can be adapted to produce single chain antibodies to immunogenicpolypeptides as described above. Accordingly, in context of the presentinvention, the term “antibody molecule” relates to full immunoglobulinmolecules as well as to parts of such immunoglobulin molecules.Furthermore, the term relates, as discussed above, to modified and/oraltered antibody molecules, like chimeric and humanized antibodies. Theterm also relates to monoclonal or polyclonal antibodies as well as torecombinantly or synthetically generated/synthesized antibodies. Theterm also relates to intact antibodies as well as to antibody fragmentsthereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab′,F(ab′)2. The term “antibody molecule” also comprises bifunctionalantibodies and antibody constructs, like single chain Fvs (scFv) orantibody-fusion proteins. It is also envisaged in context of thisinvention that the term “antibody” comprises antibody constructs whichmay be expressed in cells, e.g. antibody constructs which may betransfected and/or transduced via, inter alia, viruses or vectors. Ofcourse, the antibody of the present invention can be coupled, linked orconjugated to detectable substances.

Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission tomographies, and nonradioactive paramagnetic metalions. The detectable substance may be coupled or conjugated eitherdirectly to an Fc portion of an antibody (or fragment thereof orindirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to an Fcportion of antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, or ⁹⁹Tc. Techniques for conjugating coupling orlinked compounds to the Fc portion are well known, see, e.g., Arnon etal., “Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,“Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.),Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, inMonoclonal Antibodies '84: Biological And Clinical Applications,Pinchera et al. (eds.), pp. 475-506 (1985); “Analyzis, Results, AndFuture Prospective Of The Therapeutic Use Of Radiolabeled Antibody InCancer Therapy”, in Monoelonal Antibodies For Cancer Detection AndTherapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), andThorpe, Immunol. Rev., 119-158.

In a preferred embodiment of the present invention, a polypeptide asdescribed herein which is used in the method of the present invention isfused at its N- and/or C-terminus to a heterologous polypeptide fordetecting methylated DNA which is preferably selected from the groupconsisting of a HA-tag, myc6-tag, FLAG-tag, STREP-tag, STREP II-tag,TAP-tag, HAT-tag, chitin-binding domain (CBD), maltose-binding protein,His6-tag, Glutathione-S-transferase (GST) tag, Intein-tag,Streptavidin-binding protein (SBP) tag and a Fc-portion of an antibody.A “tag” is an amino acid sequence which is homologous or heterologous toan amino acid sequence sequence to which it is fused. Said tag may,inter alia, facilitate purification of a protein or facilitate detectionof said protein to which it is fused. The fusion refers to a co-linearlinkage and results in a translation fusion. In an also furtherpreferred embodiment a polypeptide of the present invention which iscapable of binding methylated DNA is fused to a heterologous polypeptideand optionally comprises an additional linker between the N- and/orC-terminus of said polypeptide and said heterologous polypeptide. Saidlinker is preferably a flexible linker. Preferably, it comprises plural,hydrophilic, peptide-bonded amino acids. Optionally, the linkercomprises a protease cleavage site which allows to cut off theheterologous polypeptide fused to a polypeptide of the presentinvention, if desirable. Protease cleavage sites are, for example, athrombin cleavage site.

Preferably, said linker comprises a plurality of glycine, alanine,aspartate, glutamate, proline, isoleucine and/or arginine residues. Itis further preferred that said polypeptide linker comprises a pluralityof consecutive copies of an amino acid sequence. Usually, thepolypeptide linker comprises 1 to 20, preferably 1 to 19, 1 to 18, 1 to17, 1 to 16 or 1 to 15 amino acids although polypeptide linkers of morethan 20 amino acids may work as well.

Preferably, said Fc protein of an antibody comprises preferably at leasta portion of the constant region of an immunoglobulin heavy chainmolecule. The Fc region is preferably limited to the constant domainhinge region and the C_(H)2 and C_(H)3 domains. The Fc region in apolypeptide of the present invention which is capable of bindingmethylated DNA and which is employed in the method of the presentinvention can also be limited to a portion of the hinge region, theportion being capable of forming intermolecular disulfide bridges, andthe C_(H)2 and C_(H)3 domains, or functional equivalents thereof.

Alternatively, it is also preferred that the Fc portion comprises atleast so many C_(H) regions which are required such that a polypeptideof the present invention capable of binding methylated DNA has still theproperties of a polypeptide described hereinabove, in particular theproperties of the polypeptide used in the appended Examples.

In another alternative, it is also preferred that said constant regionmay contain one or more amino acid substitutions when compared toconstant regions known in the art. Preferably it contains 1 to 100, 1 to90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30 or 1 to 20,more preferably 1 to 10, even more preferably 1 to 9, 1 to 8, 1 to 7 or1 to 6 and most preferably 1 to 5, 1 to 4, 1 to 3 or 2 or 1substitution(s). The comparison is preferably done as is known in theart or, more preferably, as described elsewhere herein.

Alternatively, said constant region comprises preferably at least theC_(H)1 region, more preferably the C_(H)1 and C_(H)2 regions and mostpreferably the C_(H)1, C_(H)2 and C_(H)3 region. As is known in the art,the constant region of an antibody contains two immunoglobulin heavychains which harbour three characteristic immunoglobulin domainscomposed of about 110 amino acids, wherein the two immunoglobulin heavychains are covalently linked via disulfide bonds.

It is also envisaged that the constant region could preferably be ofchicken or duck origin. Yet, preferably, the constant region is of theIgM, IgA, IgD or IgE isotype and more preferably it is of the IgGisotype, most preferably of the IgG1 isotype. Preferably, theaforementioned isotypes are of vertebrate origin, more preferably ofmammal origin, even more preferably of mouse, rat, goat, horse, donkey,camel or chimpanzee origin and most preferably of human origin.Preferably, said IgG isotype is of class IgG1, IgG2, IgG3, IgG4 and saidIgA isotype is of class IgA1, IgA2.

In a further preferred embodiment of the present invention a polypeptideused in the method of the present invention is a fusion protein betweenthe methyl-DNA binding domain of the MBD2 protein and the Fc portion ofan antibody as disclosed herein. Optionally the preferred fusion proteincomprises a linker polypeptide as described herein, wherein said linkerpolypeptide is preferably located between the methyl-DNA binding domainof MBD2 and the Fc portion of an antibody.

The herein described heterologous polypeptide fused to a polypeptideused in the method of the present invention facilitates binding and/orattachment of a polypeptide used in the method of the present inventionsto a container or solid support including, but not limited to, glass,cellulose, polyacrylamide, nylon, polycarbonate, polystyrene, polyvinylchloride or polypropylene or the like. Preferably, said container is aPCR-tube composed of polycarbonate and more preferably it is a heatstable TopYield™ strip from Nunc Cat. No. 248909. Said PCR-tube or stripmay be in the format of a 96-well, 384-well or 1024-well plate.Accordingly, the method of the present invention is suitable forhigh-through put applications which can be automated since the method ofthe present invention can be performed as so-called “one tube—oneassay”.

In a preferred embodiment, the container or solid support, preferably aPCR-tube or stripe is coated directly or indirectly with a polypeptideused in the method of the present invention: for example, coating wouldbe achieved directly by using a biotinylated polypeptide of the presentinvention and a streptavidin coated container, preferably a PCR-tube.However, any other technique known in the art for coating a containerwith a polypeptide are contemplated by the present invention. Indirectcoating can preferably be achieved by an antibody coated onto thesurface of said container and which is capable to specifically bindeither a polypeptide of the present invention which is capable ofbinding methylated DNA or specifically binding the heterologouspolypeptide preferably fused to said polypeptide capable of bindingmethylated DNA or specifically binding the anti-methylated DNA antibodyof the present invention. In fact, said container is indirectly coatedwith a polypeptide of the present invention which is capable of bindingmethylated DNA.

Coating of the container as described herein may be achieved, forexample, by coating said container with an agent which is suitable tointeract with the heterologous polypeptide fused to a polypeptide of thepresent invention which is capable of binding methylated DNA. Forexample, said container may be coated with glutathione and, accordingly,a GST-tagged polypeptide of the present invention is bound byglutathione which results in coating of said container with apolypeptide to be employed in the method of the present invention.Preferably, coating of the container occurs due to the property of theplastic out of which the preferred container described herein is built.Accordingly, when a polypeptide of the present invention is brought incontact with a container of the present invention, said polypeptidecoates said container.

As mentioned herein, the method of the present invention allows thedetection of methylated DNA of, preferably, a single gene locus whichrenders it a suitable diagnostic tool for, inter alia, detectingmethylated DNA from more than 15 μg, less than 15 μg, less than 10 μg,less than 10 ng, 7.5 ng, 5 ng, 2.5 ng, 1 ng, 0.5 ng, 0.25 ng, or about150 pg. By the term biological sample obtained from a subject or anindividual, cell line, tissue culture, or other source containingpolynucleotides or polypeptides or portions thereof. As indicated,biological samples include body fluids (such as blood, sera, plasma,urine, synovial fluid and spinal fluid) and tissue sources found toexpress the polynucleotides of the present invention. Methods forobtaining tissue biopsies and body fluids from mammals are well known inthe art. A biological sample which includes genomic DNA, mRNA orproteins is preferred as a source.

Without being bound by theory, it is believed that methylation of CpGdinucleotides correlates with stable transcriptional repression andpresumably leads to the fact that large parts of the non-coding genomeand potentially harmful sequences are not transcribed. A global DNAhypomethylation has been described for almost all kinds of tumours. Intumour tissue, the content in 5-methylcytosine is reduced compared tonormal tissue with the major share of demethylation events being foundin repetitive satellite sequences or in centromer regions of thechromosomes. However, in single cases, the demethylation and activationof proto-oncogenes such as, e.g., bcl-2 or c-myc have also beendescribed (Costello, J. Med. Genet. 38 (2001), 285-303). CpG islands ingeneral exert gene regulatory functions. This is why a change in thestatus of methylation correlates mostly directly with a change in thetranscriptional activity of the locus concerned (Robertson (1999);Herman (2003); Esteller (2002); Momparler (2003); Plass (2002), all loc.cit.). Most CpG islands are present in unmethylated form in normalcells. In certain situations, CpG islands can, however, also bemethylated in gene regulatory events. The majority of CpG islands of theinactivated X-chromosome of a female cell are, for example, methylated(Goto, Microbiol. Mol. Biol. Rev. 62 (1998), 362-378). CpG islands canbe methylated also in the course of normal aging processes (Issa, Clin.Immunol. 109 (2003), 103-108).

It is in particular in tumours that CpG islands which are normally notmethylated can be present in a hypermethylated form. In many cases,genes affected by the hypermethylation encode proteins which counteractthe growth of a tumour such as, e.g., tumour suppressor genes. Examplesof genes for which it could be shown that they can be inactivated intumours through the epigenetic mechanism of hypermethylation aredescribed herein above. Reasons for the tumour-specific hypermethylationare almost unknown. Interestingly, certain kinds of tumours seem to havetheir own hypermethylation profiles. It could be shown in largercomparative studies that hypermethylation is not evenly distributed butthat it occurs depending on the tumour. In cases of leukaemia, mostlyother genes are hypermethylated compared to, for instance, coloncarcinomas or gliomas. Thus, hypermethylation could be useful forclassifying tumours (Esteller, Cancer Res. 61 (2001), 3225-3229;Costello, Nat. Genet. 24 (2000), 132-138).

Thus, it is believed that epigenetic effects such as hypo- and/orhypermethylation are correlated with cancers, tumors and/or metastatis.

The subject of the present invention from which the sample is obtainedfor detecting methylated DNA is suspected to have hypo- and/orhypermethylated genloci. Said hypo- and/or hypermethylated genloci areindicative of a cancer, tumor or metastasis. The tumor or cancer can beany possible type of tumor or cancer. Examples are skin, breast, brain,cervical carcinomas, testicular carcinomas, head and neck, lung,mediastinum, gastrointestinal tract, genitourinary system,gynaecological system, breast, endocrine system, skin, childhood,unknown primary site or metastatic cancer, a sarcoma of the soft tissueand bone, a mesothelioma, a melanoma, a neoplasm of the central nervoussystem, a lymphoma, a leukaemia, a paraneoplastic syndrome, a peritonealcarcinomastosis, a immunosuppression-related malignancy and/ormetastatic cancer etc. The tumor cells may, e.g., be derived from: headand neck, comprising tumors of the nasal cavity, paranasal sinuses,nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivaryglands and paragangliomas, a cancer of the lung, comprising non-smallcell lung cancer, small cell lung cancer, a cancer of the mediastinum, acancer of the gastrointestinal tract, comprising cancer of theoesophagus, stomach, pancreas, liver, biliary tree, small intestine,colon, rectum and anal region, a cancer of the genitourinary system,comprising cancer of the kidney, urethra, bladder, prostate, urethra,penis and testis, a gynaecologic cancer, comprising cancer of thecervix, vagina, vulva, uterine body, gestational trophoblastic diseases,ovarian, fallopian tube, peritoneal, a cancer of the breast, a cancer ofthe endocrine system, comprising a tumor of the thyroid, parathyroid,adrenal cortex, pancreatic endocrine tumors, carcinoid tumor andcarcinoid syndrome, multiple endocrine neoplasias, a sarcoma of the softtissue and bone, a mesothelioma, a cancer of the skin, a melanoma,comprising cutaneous melanomas and intraocular melanomas, a neoplasm ofthe central nervous system, a cancer of the childhood, comprisingretinoblastoma, Wilm's tumor, neurofibromatoses, neuroblastoma, Ewing'ssarcoma family of tumors, rhabdomyosarcoma, a lymphoma, comprisingnon-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary centralnervous system lymphoma, and Hodgkin's disease, a leukaemia, comprisingacute leukemias, chronic myelogenous and lymphocytic leukemias, plasmacell neoplasms and myelodysplastic syndromes, a paraneoplastic syndrome,a cancer of unknown primary site, a peritoneal carcinomastosis, aimmunosuppression-related malignancy, comprising AIDS-relatedmalignancies, comprising Kaposi's sarcoma, AIDS-associated lymphomas,AIDS-associated primary central nervous system lymphoma, AIDS-associatedHodgkin's disease and AIDS-associated anogenital cancers, andtransplantation-related malignancies, a metastatic cancer to the liver,metastatic cancer to the bone, malignant pleural and pericardialeffusions and malignant ascites. It is mostly preferred that said canceror tumorous disease is cancer of the head and neck, lung, mediastinum,gastrointestinal tract, genitourinary system, gynaecological system,breast, endocrine system, skin, childhood, unknown primary site ormetastatic cancer, a sarcoma of the soft tissue and bone, amesothelioma, a melanoma, a neoplasm of the central nervous system, alymphoma, a leukemia, a paraneoplastic syndrome, a peritonealcarcinomastosis, a immunosuppression-related malignancy and/ormetastatic cancer. Preferred tumors are AML, plasmacytoma or CLL.

As mentioned herein, the present invention provides a method fordetecting methylated DNA, preferably CpG-methylated DNA fragments in asingle-tube assay comprising the following steps: binding of genomic DNAto polypeptide which is capable of binding methylated DNA, preferably amethyl-CpG-binding protein, coated onto to the inner surface of acontainer, preferably a PCR-tube, washing off unbound (unmethylated)DNA-fragments and preferably directly applying gene-specific PCR todetect the enrichment of methylated DNA. Since the method of the presentinvention is robust, fast and is an easy applicable and reliablediagnostic tool for detecting methylated DNA due to the “one reactioncontainer for all steps”, the method of the present invention may beapplicable to high through put formats which may be made subject ofautomation. The method of the present invention allows thus an easy andhighly sensitive detection of CpG methylation of preferably (a) singlegene locus/loci. Since methylation patterns of tumors and/or cancersappear to develop into a valuable diagnostic parameter, it is preferredto provide a kit comprising all means for carrying out the method of thepresent invention.

Accordingly, the present invention relates to a kit comprising fordetecting methylated DNA according to the method of the presentinvention comprising

(a) a polypeptide capable of binding methylated DNA as described herein;(b) a container which can be coated with said polypeptide; and(c) means for coating said container; and(d) means for detecting methylated DNA.

The embodiments disclosed in connection with the method of the presentinvention apply, mutatis mutandis, to the kit of the present invention.

Advantageously, the kit of the present invention further comprises,optionally (a) reaction buffer(s), storage solutions, wash solutionsand/or remaining reagents or materials required for the conduction ofscientific or diagnostic assays or the like as described herein.Furthermore, parts of the kit of the invention can be packagedindividually in vials or bottles or in combination in containers ormulticontainer units. The kit of the present invention may beadvantageously used, inter alia, for carrying out the method fordetecting methylated DNA as described herein and/or it could be employedin a variety of applications referred herein, e.g., as diagnostic kits,as research tools or therapeutic tools. Additionally, the kit of theinvention may contain means for detection suitable for scientific,medical and/or diagnostic purposes. The manufacture of the kits followspreferably standard procedures which are known to the person skilled inthe art. The kit of the present invention is preferably useful in a“single-tube” assay as provided herein.

“Means for coating” of the container of the present invention are allagents suitable for coating said container with a polypeptide of thepresent invention, for example, cross-linking agents or avidin orglutathione or the like. Thus, basically, every agent which is suitableto interact with the heterologous polypeptide fused to a polypeptide ofthe present invention which is capable of binding methylated DNA.Preferably, the kit of the present invention comprises pre-coatedcontainers, preferably PCR-tubes.

The term “means for detecting methylated DNA” encompasses all agentsnecessary to carry out the detection methods for methylated DNA asdescribed herein above. In a more preferred embodiment, said kitcomprises an instruction manual how to carry out detection of methylatedDNA according to the method of the present invention.

The figures show:

FIG. 1: Outline of Methyl-binding (MB)-PCR. (A) The major steps of theMB-PCR procedure are illustrated. MB-PCR comprises of two separatereactions, the control-PCR reaction (P-reaction) which amplifies acandidate locus directly from a genomic template, and themethyl-CpG-binding-PCR reaction which amplifies the candidate locus fromthe template DNA that was previously bound by a methyl-CpG-bindingpolypeptide in the reaction vessel (M-reaction). In the first step, theinner walls of both reaction vessels are coated with a methyl-bindingpolypeptide and subsequently saturated using blocking reagents (step 2).The template DNA (genomic DNA restricted with Mse I or similar enzymes)is then added to one tube (M-reaction) and allowed to bind (step 3). Inthe last step, the PCR reaction mix is added directly into both tubesand 50% of template DNA previously used for the M-reaction is added tothe P-reaction. After gene-specific PCR, products may be analyzed, e.g.by agarose gel electrophoresis. The term “CpG-methylation low” used inFIGS. 1A and B comprises and particularly refers to unmethylated DNA (B)Schematic representation of the MB-PCR procedure using a recombinantmethyl-binding polypeptide MBD-Fc described herein above.

FIG. 2: Detecting CpG methylation in leukaemia cell lines at threeCpG-island promoters by MB-PCR. (A) Shown are: the position ofCpG-dinucleotides, Mse I-restriction sites, first exons and positions ofprimers used to detect promoter fragments of ICSBP, ESR1, and CDKN2B(p15^(INK4b)). (B) Representative MB-PCR results of the indicatedpromoters for eight different leukaemia cell lines. The P-reactiondirectly amplifies the genomic DNA, whereas the M-reaction onlyamplifies CpG-methylated DNA fragments.

FIG. 3: Methylation of the ICSBP promoter inversely correlates withICSBP expression in leukaemia cell lines. (A) Transcription levels ofICSBP were determined by LightCycler real time PCR relative to thehousekeeping gene ACTB. (B) U937 cells, treated with Decitabine (DAC)for the indicated time periods were analyzed for ICSBP expression.Results were normalized to ACTB expression. Data represent meanvalues±SD of two independent LightCycler analyses.

FIG. 4: Detection of aberrant CpG methylation in AML cells.Representative MB-PCRs for ESR1, CDKN2B (p15^(INK4b)), and ICSBPpromoters of several healthy donors and AML patients.

FIG. 5: MB-PCR of the ICSBP promoter correlates with the resultsobtained by bisulfite sequencing. Genomic DNA derived from cell lines aswell as cells of selected healthy donors and AML patients was treatedwith bisulfite. The indicated region of the ICSBP-gene was amplified andcloned. Several independent inserts were sequenced and results arepresented schematically. Circles mark the position of CpG-dinucleotides(empty: unmethylated; filled: methylated).

FIG. 6: Sensitivity of MB-PCR. (A) MB-PCRs for ESR1, CDKN2B(p15^(INK4b)), and ICSBP promoters from mixtures of DNA from a healthydonor (unmethylated) DNA and DNA from the cell line KG-1 (methylated inall three loci). (B) DNA from three cell lines was subjected to MB-PCRusing the indicated amounts of DNA for the M-reaction (or half of theindicated amount for the P-reaction). With decreasing amounts of DNA,the number of amplification cycles during PCR (given in parenthesis) wasincreased. Also shown is a sample that did not include DNA (H₂O).

FIG. 7: FIG. 7 shows the nucleotide sequence of plasmid pMTBip/MBD2-Fcand the protein sequence (in bold) of the MBD2-Fc bifunctional proteinwhich is encoded by plasmid pMTBip/MBD2-Fc.

The amino acid sequence of the MBD2-Fc bifunctional protein has thefollowing features.

-   -   AA 1-28 (nt 851-934): Drosophila BiP secretion signal (leader        peptide from pMT/Bip/V5-His vector):    -   AA 29-115 (nt 935-1196): AA 144-230 of human MBD2    -   AA 116-129 (nt 1196-1237): Flexible Linker (AAADPIEGRGGGGG)    -   AA 130-361 (1238-1933): AA99-330 of human IGHG1

FIG. 8: MB-PCR detects methylation of CpG-island promoters (A) Schematicpresentation of the detected MseI-fragments (indicated as grey boxes) ofESR1, CDKN2B (p15INK4b), ICSBP, ETV3 and DDX20. The position ofCpG-dinucleotides, MseI-restriction sites, transcription start site,first exon and relative position of primers are marked. (B)

Shown are representative MB-PCR results of normal (unmethylated) and invitro methylated genomic DNA for the indicated promoters. The P-reactiondirectly amplifies the genomic DNA, whereas the M-reaction onlyamplifies CpGmethylated DNA fragments.

FIG. 9: Detecting CpG methylation in leukaemia cell lines by MB-PCR. (A)Shown are representative MB-PCR results of eight different leukaemiacell lines for the indicated promoters. (B) Genomic DNA from the samecell lines was analyzed by bisulfite sequencing. The indicated region ofthe ICSBP gene was amplified and cloned. Several independent insertswere sequenced and results are presented schematically. Squares mark theposition of CpG-dinucleotides (empty: unmethylated; filled: methylated).

FIG. 10: Detection of aberrant CpG methylation in primary AML blasts.Two for the ICSBP promoter of one representative healthy donor (N) andnine AML patients are shown together with corresponding sequencingresults. (Results of bisulfite sequencing are presented as described inFIG. 9.)

A better understanding of the present invention and of its manyadvantages will be seen from the following examples, offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way.

EXAMPLE 1 Single-Tube Assay for the Detection of CpG-MethylatedDNA-Fragments Using Methyl-Binding Polymerase Chain Reaction (MB-PCR)

This method uses an approach similar to ELISAs. A protein with highaffinity for CpG-methylated DNA is coated onto the walls of a PCR-cyclercompatible reaction vessel and used to selectively capture stronglymethylated DNA-fragments from a genomic DNA mixture. The retention of aspecific DNA-fragment (e.g. a CpG island promoter of a specific gene)can be detected in the same tube using PCR (either standard PCR orrealtime PCR, single or multiplex). The degree of methylation may beestimated relative to a PCR reaction of the genomic input DNA. FIG. 1shows a schematic representation of MB-PCR.

1. Cells, Patient Samples, DNA Preparation and Fragmentation

Cells

Peripheral blood mononuclear cells (MNC) were separated by leukapheresisof healthy donors, followed by density gradient centrifugation overFicoll/Hypaque. Monocytes were isolated from MNC by countercurrentcentrifugal elutriation in a J6ME centrifuge (Beckman, München, Germany)as described in Krause, J. Leukoc. Biol. 60 (1996), 540-545. DrosophilaS2 cells were obtained from ATTC and cultured in Insect-Xpress medium(Bio Whittaker) containing 10% fetal calf serum (FCS; PAA) in anincubator at 21° C. The human myeloid leukaemia cell lines THP-1, NB-4,KG-1, K562, HL-60 and U937 were grown in RPMI 1640 medium supplementedwith 10% FCS. The human myeloid leukaemia cell line Mono Mac 6 was grownRPMI 1640 medium plus 10% FCS and 1% OPI media supplement (Sigma). Thehuman myeloid leukaemia cell line MUTZ-3 was maintained in αMEM plus 20%FCS and 10 ng/ml stem cell factor. For DNA-demethylation, U937 cellswere treated with the indicated amounts of Decitabine(2-deoxy-5′-azacytidine, Sigma) for several days.

Patient Samples

Fresh peripheral blood samples and bone marrow specimens from 35patients with newly diagnosed and untreated de novo or secondary AMLwere used for the study. All patients were treated according to theprotocol AMLCG-2000 of the German AML Cooperative Group. The study wasapproved by the Institutional Ethics Committee, and written informedconsent was obtained from each patient before entering the study.

DNA Preparation and Fragmentation

Genomic DNA from various cellular sources, including the cell linesdescribed herein (e.g. KG1, U937, and THP-1), normal human monocytes(healthy donor) and frozen blast cells from a patient with AML wereprepared using Blood and Cell Culture Midi Kit (Qiagen). Quality of thegenomic DNA-preparation was controlled by agarose gel electrophoresisand DNA concentration was determined by UV spectrophotometry. GenomicDNA was digested with Mse I (NEB) and finally quantified using PicoGreendsDNA Quantitation Reagent (Molecular Probes). Where indicated. DNA wasin vitro methylated using Sss I methylase (NEB).

2. Generation of a Recombinant Methyl-CpG-Binding Polypeptide

A cDNA corresponding to the methyl-CpG binding domain (MBD) of humanMBD2 (Genbank acc. no. NM_(—)003927; AA 144-230) was PCR-amplified fromreverse transcribed human primary macrophage total RNA using primersMBD2-Nhe_S (5′-AGA TGC TAG CAC GGA GAG CGG GAA GAG G-3′) (SEQ ID NO: 4)and MBD2-Not_AS (5′-ATC ACG CGG CCG CCA GAG GAT CGT TTC GCA GTC TC-3′)(SEQ ID NO: 5) and Herculase DNA Polymerase (Stratagene). Cyclingparameters were: 95° C., 3 min denaturation; 95° C., 20 s, 65° C., 20 s,72° C., 80s amplification for 34 cycles; 72° C., 5 min final extension.The PCR-product was precipitated, digested with Not I/Nhe I, cloned intoNotI/NheI-sites of Signal pIg plus vector (Ingenius, R&D Systems) andsequence verified resulting in pIg/MBD2-Fc (eukaryotic expressionvector). To clone pMTBip/MBD2-Fc for recombinant expression inDrosophila S2 cells, the Apa I/Nhe I-fragment of pIg/MBD2-Fc containingthe MBD of human MBD2 fused to the Fc-tail of human IgG1 was subclonedinto Apa I/Spe I-sites of pMTBiP/V5-His B (Invitrogen).

Drosophila S2 cells were obtained from ATTC and cultured inInsect-Xpress medium (Bio Whittaker) containing 10% FCS (PM) in anincubator at 21° C. 4×10⁶ Drosophila S2 cells/60 mm cell culture dishwere transfected with a mixture of 1.5 μg pMTBip/MBD2-Fc and 0.3 μgpCoHygro (Invitrogen) using Effectene transfection reagent (Qiagen)according to the manufacturers protocol. On day three, transfected cellswere harvested, washed and replated in selection medium (Insect-Xpress)containing 10% FCS and 300 μg/ml Hygromycin (BD Biosciences). Selectionmedium was replaced every 4-5 days for five weeks. The pool of stablytransfected Drosophila S2 cells was expanded. For large scale productionof the methyl-CpG binding polypeptide MBD-Fc, 1−5×10⁸ cells werecultured in 100-200 ml Insect-Xpress without FCS (optional: 300 μg/mlHygromycin) in 2000 ml roller bottles for two days before the additionof 0.5 mM CuSO₄. Medium was harvested every 4-7 days and cells werereplated medium plus CuSO₄ for further protein production. Cell culturesupernatants were combined, dialysed against TBS (pH 7.4) and purifiedusing a protein A column. The MBD-Fc containing fractions were combinedand dialysed against TBS (pH 7.4). The stably transfected Drosophila S2cells produced 3-5 mg recombinant MBD2-Fc protein per litre cell culturesupernatant. The sequence and features of the MBD-Fc protein are shownin FIG. 7.

3. Preparation of MB-PCR Tubes

50 μl of the recombinant MBD2-Fc protein comprising the methyl-CpGbinding domain (MBD) of human methyl-CpG-binding domain 2 (MBD2), aflexible linker polypeptide and the Fc portion of human IgG1 (diluted at15 μg/ml in 10 mM Tris/HCl pH 7.5) were added to each well of heatstable TopYield™ Strips (Nunc Cat. No. 248909) and incubated overnightat 4° C. Wells were washed three times with 200 μl TBS (20 mM Tris, pH7.4 containing 170 mM NaCl) and blocked at RT for 3-4 h with 100 μlBlocking Solution (10 mM Tris, pH 7.5 containing 170 mM NaCl, 5% skimmilk powder, 5 mM EDTA and 1 μg/ml of each poly d(I/C), poly d(A/T) andpoly d(CG), all from Amersham). Tubes were washed three times with 200μl TBST (TBS containing 0.05% Tween-20).

4. Binding of Methylated DNA Fragments

50 μl Binding Buffer (20 mM Tris, pH 7.5 containing 400 mM NaCl, 2 mMMgCl₂, 0.5 mM EDTA, and 0.05% Tween-20) were added to each well and 2 μlMse I-digested DNA (5 ng/μl) was added to every second well(M-reaction). Wells were incubated on a shaker at RT for 40-50 min.Tubes were washed two times with 200 μl Binding Buffer and once with 10mM Tris/HCl pH 8.0.

5. Detection of Methylated DNA Fragments

PCR was carried out directly in the treated and washed TopYield™ Strips.The PCR-mix (PCR Master Mix (Promega); 50 μl-reactions/well) included 10pmol of each gene-specific primer (synthesized by Metabion). Primersequences were P15 S (5′-GGC TCA GCT TCA TTA CCC TCC-3′) (SEQ ID NO: 6),P15 AS (5′-AAA GCC CGG AGC TAA CGA C-3′) (SEQ ID NO: 7), ESR1S (5′-GACTGC ACT TGC TCC CGT C-3′) (SEQ ID NO: 8), ESR1 AS (5′-AAG AGC ACA GCCCGA GGT TAG-3′) (SEQ ID NO: 9), ICSBP S (5′-CGG AAT TCC TGG GAA AGCC-3′) (SEQ ID NO: 10), ICSBP AS (5′-TTC CGA GAA ATC ACT TTC CCG-3′) (SEQID NO: 11), METS S (5′-AAT TGC GTC TGA AGT CTG CGG-3′), (SEQ ID NO. 12),METS AS (5′-TCC CAC ACA ACA GAG AGG CG-3′) (SEQ ID NO. 13), DP103 S(5′-GCT GTT AGT CCA GTT CCA GGT TCC-3′) (SEQ ID NO. 14), DP103 AS(5′-GTG CM CCA CAT TTA TCT CCG G-3′) (SEQ ID NO: 15).

After adding the PCR-mix, 1 μl Mse I-digested DNA (5 ng/μl) was added toevery second other well, that was not previously incubated withDNA-fragments (P-reaction). PCR was performed on a MJResearch enginewith the following cycling conditions: 95° C. for 3 min (denaturation),94° C. for 20 s, 60° C. for 20 s and 72° C. for 70 s (36 cycles) and 72°C. for 5 min (final extension). PCR-products were analyzed using 3%agarose gel electrophoresis and the ethidium bromide stained gel wasscanned using a Typhoon 9200 Imager (Amersham/Pharmacia).

6. Sodium Bisulfite Sequencing

Modification of DNA with sodium bisulfite was performed as previouslydescribed. Bisulfite-treated DNA was amplified in a nested PCR reactionusing the primers icsbp-out S (5′-GGG GTA GTT AGT TTT TGG TTG-3′) (SEQID NO: 16) and icsbp-out AS (5′-ATA MT AAT TCC ACC CCC AC-3′) (SEQ IDNO: 17) for the first and icsbp-in S (5′-TTG TGG ATT TTG ATT MT GGG-3′)(SEQ ID NO: 18) and icsbp-in AS (5′-CCR CCC ACT ATA CCT ACC TAC C-3′)(SEQ ID NO: 19) for the second round of amplification. PCR-products werecloned using TOPO-TA Cloning Kit (Invitrogen) and several independentclones were sequenced.

7. RNA-Preparation, Real-time-PCR

Total RNA was isolated from different cell lines by the guanidinethiocyanate/acid phenol method (Chomczynski, Anal. Biochem. 162 (1987),156-159. RNA (2 μg) was reverse transcribed using Superscript II MMLV-RT(Invitrogen). Real-time PCR was performed on a Lightcycler (Roche) usingthe Quantitect kit (Qiagen) according to the manufacturer'sinstructions. Primers used were: human ICSBP: sense 5′-CGT GGT GTG CMAGG CAG-3′ (SEQ ID NO: 20), antisense 5′-CTG TTA TAG AAC TGC TGC AGC TCTC-3′ (SEQ ID NO: 21); human ACTB (β-Actin): sense 5′-TGA CGG GGT TCA CCCACA CTG TGC CCA TCT A-3′ (SEQ ID NO: 22), antisense 5′-CTA GM GCA TTTGTG GTG GAC GAT GGA GGG-3′ (SEQ ID NO: 23). Cycling parameters were:denaturation 95° C., 15 min, amplification 95° C., 15 s, 57° C., 20 s,72° C., 25 s, for 50 cycles. The product size was initially controlledby agarose gel electrophoresis and melting curves were analyzed tocontrol for specificity of the PCR reactions. ICSBP data were normalizedfor expression of the housekeeping gene β-actin (ACTB). The relativeunits were calculated from a standard curve plotting 3 differentconcentrations of log dilutions against the PCR cycle number (CP) atwhich the measured fluorescence intensity reaches a fixed value. Theamplification efficiency E was calculated from the slope of the standardcurve by the formula: E=10^(−1/slope). E_(ICSBP) was in the range of1.87 to 1.98, E_(ACTB) ranged from 1.76 to 1.84. For each sample, dataof 3 independent analyzes were averaged.

8. Analyzing the CpG Island Methylation Status of ESR1, CDKN2B(p15^(INK4b)), and ICSBP Promoters by MB-PCR

Several leukaemia cell lines were analyzed for their CpG islandmethylation status of ESR1, CDKN2B (p15^(INK4b)), and ICSBP promoters byMB-PCR. Genomic DNA was digested with Mse I. This enzyme was chosenbecause it is methylation-insensitive and cuts DNA into small fragmentsbut leaves CpG islands relatively intact. Location of the gene-specificMse I-fragments relative to the first intron of their respective genesas well as positions of gene-specific primers used for PCR are shown inFIG. 2A. All fragments were chosen to include the putative proximalpromoter regions. A total of 10 ng of restricted DNA were used for theM-reaction and 5 ng of the same digested genomic DNA were used for theP-reaction. The result of a representative MB-PCR experiment from eightdifferent leukaemia cell lines is shown in FIG. 2B. The ESR1 promoterwas amplified to varying degrees in the M-reaction of all eight samples,which is in line with previous reports demonstrating its aberrantmethylation in 86% of human haematopoietic tumours. The P-reaction forthe CDKN2B (p15^(INK4b)) promoter failed completely in three cell lines(THP-1, NB-4, K562) suggesting mutations or deletions on both alleles,which has also been demonstrated before. Two cell lines (KG-1 and MUTZ3)showed a positive M-reaction for the CDKN2B (p15^(INK4b)) promoter,whereas three cell lines (U937, MonoMac6, HL-60) were negative. Theobserved results were in good concordance with previously publishedmethylation analyzes of ESR1 and CDKN2B (p15^(INK4b)) promoters in someof these cell lines. In some cases, P-reactions were weaker incomparison with other cell types, suggesting the loss or mutation of oneallele (e.g. ESR1 in U937 cells). The ICSBP promoter was also amplifiedin M-reactions of six cell lines.

The degree and effect of ICSBP promoter methylation was analyzed tofurther validate the experimental potential of MB-PCR. Expression levelsof ICSBP were analyzed in the eight leukaemia cell lines usingLightCycler Real time PCR. As shown in FIG. 3A, mRNA expression levelsinversely correlated with methylation degree as determined by MB-PCR.Treatment of U937 cells, which show a high degree of ICSBP promotermethylation with the demethylating agent Decitabine(5-Aza-2′Deoxycytidine) led to a marked, dose- and time-dependentinduction of ICSBP mRNA expression (s. FIG. 3B), indicating that themethylation-induced repression of ICSBP transcription is reversible inthese cells.

To test whether MB-PCR is also able to detect the methylation of CpGisland promoters in primary tumour cells, DNA was prepared from bloodmonocytes of healthy individuals (n=4) and blast cells of patients withAML (n=11), digested with Mse I, and subjected to MB-PCR. As shown inFIG. 4, no significant level of methylation was detected in the DNA ofhealthy donors, whereas most patients showed significant methylation inat least one of the three promoters analyzed.

To determine how MB-PCR results correlate with the exact pattern of CpGmethylation at the ICSBP promoter, ICSBP promoter methylation wasanalyzed by bisulfite sequencing in selected cell lines, normal andtumour cells. The results shown in FIG. 5 indicate that the degree ofpromoter methylation can be predicted by MB-PCR—strong amplificationsignals appear to indicate a high degree, whereas weaker signalsindicate a lesser degree of methylation.

Since patient samples may be contaminated with normal, potentiallyunmethylated cells, the effect of increasing amounts of normal DNA in aDNA sample of a tumor cell line was determined. Restricted DNA was mixedand subjected to MB-PCR. The results are shown in FIG. 6A. The signal inthe M-reaction decreased in a linear fashion with increasing amounts ofnormal, unmethylated DNA in the sample. To test the sensitivity of themethod, MB-PCR experiments using decreasing amounts of DNA wereperformed. As shown in FIG. 6B, comparable results were obtained usingall concentrations tested (10 ng-160 pg) when analyzing the methylationstatus of the ICSBP locus in three different cell lines. These resultsindicate, that MB-PCR can detect methylated DNA-fragments in mixtures ofnormal and tumour cells and works within the normal sensitivity range ofstandard genomic PCR (down to 160 pg of DNA).

9. Analyzing the CpG Island Methylation Status of ESR1, CDKN2B(p15^(INK4b)), ICSBP, ETV3 and DDX20 Promoters by MB-PCR,

In another experiment, the MB-PCR method was explored by analyzing thedegree of CpG methylation of single CpG island promoters that werepreviously shown to be frequently methylated in leukaemia cells, namelythe human CDKN2B gene (also known as p15_(INK4b)) and the human estrogenreceptor 1 (ESR1) gene. In addition to the well established tumormarkers three additional genes with CpG island promoters that couldpotentially act as tumor suppressor genes were selected: the humaninterferon consensus binding protein (ICSBP) gene, the human Ets variant3 gene (ETV3), and the human DEAD box polypeptide 20 gene (DDX20).ICSBP, a transcription factor of the interferon (IFN) regulatory factorfamily (IRF), is frequently down-regulated in human myeloid leukaemia(Schmidt, Blood 91 (1991), 22-29) and ICSBP-deficient mice displayhematological alterations similar to chronic myelogenous leukaemia (CML)in humans (Holtschke, Cell 87 (1996), 307-317), suggesting a tumorsuppressor function for ICSBP in hemopoietic cells. In mice, the Etsrepressor ETV3 (also known as METS or PEI) and its co-repressor DDX20(also known as DP103) were shown to link terminal monocyticdifferentiation to cell cycle arrest (Klappacher, Cell 109 (2002),169-180), which may also indicate a possible tumor suppressor role. As avalidation of our approach, genomic DNA from normal cells was eitherleft untreated or methylated in vitro using SssI, digested with MseI andsubjected to MB-PCR. Genomic DNA was digested with MseI because thisenzyme is methylation-insensitive and cuts DNA into small fragmentswhile leaving CpG islands relatively intact (Cross, Nat. Genet. 6(1994), 236-244). Locations of the gene-specific MseI-fragments relativeto the first intron of their respective genes as well as positions ofgene-specific primers used for MB-PCR are shown in FIG. 8A. Allfragments include the putative proximal promoter regions. As shown inFIG. 8B, the M-reactions of all five loci were negative when normal DNAwas used, indicating that these genomic regions are, as expected, freeof methylation in normal blood cells. However, each locus was amplifiedin the corresponding M-reaction when the same DNA was in vitromethylated using SssI-methylase before it was subjected to MB-PCR.Hence, MB-PCR is able to discriminate the methylated and unmethylatedstate at these loci.

10. Methylation Status of Specific CpG Island Promoters in Tumour CellLines Analyzed by MB-PCR.

In another experiment it was tested whether MB-PCR is able to detect themethylation status of the above loci in biological samples, severalleukaemia cell lines were analyzed. Routinely, a total of 10 ng ofrestricted DNA was used for the M-reaction and 5 ng of the same digestedgenomic DNA was used for the P-reaction. The result of a representativeMB-PCR experiment from eight different leukaemia cell lines is shown inFIG. 9A. The ESR1 promoter was amplified to varying degrees in theM-reaction of all eight samples, which is in line with previous reportsdemonstrating its aberrant methylation in more than 80% of humanhemopoietic tumors. The P-reaction for the CDKN2B promoter failedcompletely in three cell lines (THP-1, NB-4, K562) suggesting mutationsor deletions on both alleles, which has been demonstrated previously inthe cases of NB-4 (Chim, Ann. Hematol. 82 (2003), 738-742) and K562(Paz, Cancer Res. 63 (2003), 1114-1121). The two cell lines KG-1 andMUTZ3 showed a positive M-reaction for the CDKN2B promoter, whereasthree cell lines (U937, MonoMac6, HL-60) were negative. The observedresults were in good concordance with previously published methylationanalyses of ESR1 (27) and CDKN2B promoters (Cameroon, Blood 94 (1999),2445-2451; Chim (2003), loc. cit.; Paz (2003), loc. cit.). In somecases, P-reactions were weaker in comparison with other cell types,suggesting the loss or mutation of one allele (e.g. ESR1 in U937 cells).Interestingly, the ICSBP promoter was also amplified in M-reactions ofsix cell lines, whereas no significant methylation was detected at thepromoters of ETV3 and DDX20 genes.

To determine how MB-PCR results correlate with the exact pattern of CpGmethylation at the ICSBP promoter in individual cell lines, the ICSBPpromoter methylation was analyzed by bisulfite sequencing. The resultsshown in FIG. 9B indicate that the degree of promoter methylationcorresponds with results obtained by MB-PCR. Strong amplificationsignals (comparable to the corresponding P-reaction) as seen in KG-1,U937, MUTZ-3, HL-60, and K562 cell lines, appear to indicate a highdegree, whereas weaker signals (as observed for NB-4 cells) indicate alesser degree of methylation. In the absence of DNA methylation (THP-1and MonoMac6 cells) the MB-PCR is negative.

11. Detecting Methylation of CpG Island Promoters in Primary TumorCells.

DNA was prepared from blood monocytes of several healthy persons (n=4)and leukaemic blasts of patients with previously untreated AML (n=35),digested with MseI, and subjected to MB-PCR. FIG. 11 showsrepresentative ICSBP MB-PCR and corresponding bisulfite sequencingresults for 9 AML patients and 1 normal individual. In general, theintensity of the band observed in the M-reaction (as compared to thecorresponding P-reaction) showed good correlation with the mean densityof methylation in the sample. Out of 35 AML-patients tested, 7 patients(20%) showed positive MB-PCR results for ICSBP, 21 patients (60%) forESR1 and 25 patients (71%) for CDKN2B (data not shown). The frequenciesfor ESR1 and CDKN2B methylation observed concur with those described inprevious studies. ICSBP methylation apparently only affects a subgroupof patients. Twelve patients were tested for methylation of ETV3 andDDX20 genes and, as observed for the leukaemia cell lines, nosignificant methylation was detected in any of the samples.

1. An in vitro method for detecting methylated DNA comprising (a)coating a container with a polypeptide capable of binding methylatedDNA; (b) contacting said polypeptide with a sample comprising methylatedand/or unmethylated DNA; and (c) detecting the binding of saidpolypeptide to methylated DNA.
 2. The method of claim 1, wherein step(c) comprises restriction enzyme digestion, bisulfite sequencing,pyrosequencing, Southern Blot or PCR.
 3. The method of claim 1, whereinstep (c) comprises PCR.
 4. The method of claim 1, further comprisingstep (d) analyzing the methylated DNA.
 5. The method of claim 4, whereinsaid analyzing said methylated DNA comprises sequencing.
 6. The methodof claim 1, wherein said polypeptide is selected from the groupconsisting of (a) a polypeptide belonging to the Methyl-DNA bindingprotein (MBD) family; (b) a fragment of the polypeptide of (a), whereinsaid fragment is capable of binding methylated DNA; (c) a variant of thepolypeptide of (a) or the fragment of (b), wherein in said variant oneor more amino acid residues are substituted compared to the polypeptideof (a) or the fragment of (b), and wherein said variant is capable ofbinding methylated DNA; (d) a polypeptide which is an anti-methylatedDNA antibody or fragment thereof; and (e) a polypeptide which is atleast 70% identical to a polypeptide of any one of (a) to (c) and whichis capable of binding methylated DNA.
 7. The method of claim 1, whereinsaid polypeptide is fused to a heterologous polypeptide.
 8. The methodof claim 7, wherein said heterologous polypeptide is selected from thegroup consisting of a HA-tag, myc6-tag, FLAG-tag, STREP-tag, STREPII-tag, TAP-tag, HAT-tag, chitin-binding domain (CBD), maltose-bindingprotein, His6-tag, Glutathione-S-transferase (GST) tag, Intein-tag,Streptavidin-binding protein (SBP) tag and a Fc-portion of an antibody.9. The method of claim 6, wherein said anti-methylated DNA antibody isan anti-5-methlycytosine antibody or a Fab, F(ab′)₂, Fv or scFv fragmentthereof.
 10. The method of claim 1, wherein said container is coateddirectly or indirectly with said polypeptide.
 11. The method of claim 1,wherein said sample is from a subject.
 12. The method of claim 11,wherein said subject is suspected to have hypo- and/or hypermethylatedgenloci.
 13. The method of claim 12, wherein said hypo- and/orhypermethylated genloci are indicative of a cancer, tumor or metastasis.14. A kit for detecting methylated DNA according to the method of claim1 comprising (a) a polypeptide capable of binding methylated DNA; (b) acontainer which can be coated with said polypeptide; and (c) means forcoating said container; and (d) means for detecting methylated DNA.